Oligonucleotide analogues

ABSTRACT

The present invention relates to novel bicyclic and tricyclic nucleoside and nucleotide analogues as well as to oligonucleotides comprising such elements. The nucleotide analogues, LNAs (Locked Nucleoside Analogues), are able to provide valuable improvements to oligonucleotides with respect to affinity and specificity towards complementary RNA and DNA oligomers. The novel type of LNA modified oligonucleotides, as well as the LNAs as such, are useful in a wide range of diagnostic applications as well as therapeutic applications. Among these can be mentioned antisense applications, PCR applications, strand displacement oligomers, as substrates for nucleic acid polymerases, as nucleotide based drugs, etc. The present invention also relates to such applications.

FIELD OF THE INVENTION

The present invention relates to the field of bi- and tricyclicnucleoside analogues and to the synthesis of such nucleoside analogueswhich are useful in the formation of synthetic oligonucleotides capableof forming nucleobase specific duplexes and triplexes with singlestranded and double stranded nucleic acids. These complexes exhibithigher thermostability than the corresponding complexes formed withnormal nucleic acids. The invention also relates to the field of bi- andtricyclic nucleoside analogues and the synthesis of such nucleosideswhich may be used as therapeutic drugs and which may be incorporated inoligonucleotides by template dependent nucleic acid polymerases.

BACKGROUND

Synthetic oligonucleotides are widely used compounds in disparate fieldssuch as molecular biology and DNA-based diagnostics and therapeutics.

Therapeutics

In therapeutics, e.g., oligonucleotides have been used successfully toblock translation in vivo of specific mRNAs thereby preventing thesynthesis of proteins which are undesired or harmful to thecell/organism. This concept of oligonucleotide mediated blocking oftranslation is known as the “antisense” approach. Mechanistically, thehybridising oligonucleotide is thought to elicit its effect by eithercreating a physical block to the translation process or by recruitingcellular enzymes that specifically degrades the mRNA part of the duplex(RNAseH).

More recently, oligoribonucleotides and oligodeoxyribonucleotides andanalogues thereof which combine RNAse catalytic activity with theability to sequence specifically interact with a complementary RNAtarget (ribozymes) have attracted much interest as antisense probes.Thus far ribozymes have been reported to be effective in cell culturesagainst both viral targets and oncogenes.

To completely prevent the synthesis of a given protein by the antisenseapproach it is necessary to block/destroy all mRNAs that encode thatparticular protein and in many cases the number of these mRNA are fairlylarge. Typically, the mRNAs that encode a particular protein aretranscribed from a single or a few genes. Hence, by targeting the gene(“antigene” approach) rather than its mRNA products it should bepossible to either block production of its cognate protein moreefficiently or to achieve a significant reduction in the amount ofoligonucleotides necessary to elicit the desired effect. To blocktranscription, the oligonucleotide must be able to hybridise sequencespecifically to double stranded DNA. In 1953 Watson and Crick showedthat deoxyribo nucleic acid (DNA) is composed of two strands (Nature,1953, 171, 737) which are held together in a helical configuration byhydrogen bonds formed between opposing complementary nucleobases in thetwo strands. The four nucleobases, commonly found in DNA are guanine(G), adenine (A), thymine (T) and cytosine (C) of which the G nucleobasepairs with C, and the A nucleobase pairs with T. In RNA the nucleobasethymine is replaced by the nucleobase uracil (U) which similarly to theT nucleobase pairs with A. The chemical groups in the nucleobases thatparticipate in standard duplex formation constitute the Watson-Crickface. In 1959, Hoogsteen showed that the purine nucleobases (G and A) inaddition to their Watson-Crick face have a Hoogsteen face that can berecognised from the outside of a duplex, and used to bind pyrimidineoligonucleotides via hydrogen bonding, thereby forming a triple helixstructure. Although making the “antigene” approach conceptually feasiblethe practical usefulness of triple helix forming oligomers is currentlylimited by several factors including the requirement for homopurinesequence motifs in the target gene and a need for unphysiologically highionic strength and low pH to stabilise the complex.

The use of oligonucleotides known as aptamers are also being activelyinvestigated. This promising new class of therapeutic oligonucleotidesare selected in vitro to specifically bind to a given target with highaffinity, such as for example ligand receptors. Their bindingcharacteristics are likely a reflection of the ability ofoligonucleotides to form three dimensional structures held together byintramolecular nucleobase pairing.

Likewise, nucleosides and nucleoside analogues have proven effective inchemotherapy of numerous viral infections and cancers.

Also, various types of double-stranded RNAs have been shown toeffectively inhibit the growth of several types of cancers.

Diagnostics

In molecular biology, oligonucleotides are routinely used for a varietyof purposes such as for example (i) as hybridisation probes in thecapture, identification and quantification of target nucleic acids (ii)as affinity probes in the purification of target nucleic acids (iii) asprimers in sequencing reactions and target amplification processes suchas the polymerase chain reaction (PCR) (iv) to clone and mutate nucleicacids and (vi) as building blocks in the assembly of macromolecularstructures.

Diagnostics utilises many of the oligonucleotide based techniquesmentioned above in particular those that lend themselves to easyautomation and facilitate reproducible results with high sensitivity.The objective in this field is to use oligonucleotide based techniquesas a means to, for example (i) tests humans, animals and food for thepresence of pathogenic micro-organisms (ii) to test for geneticpredisposition to a disease (iii) to identify inherited and acquiredgenetic disorders, (iv) to link biological deposits to suspects in crimetrials and (v) to validate the presence of micro-organisms involved inthe production of foods and beverages.

General Considerations

To be useful in the extensive range of different applications outlinedabove, oligonucleotides have to satisfy a large number of differentrequirements. In antisense therapeutics, for instance, a usefuloligonucleotide must be able to penetrate the cell membrane, have goodresistance to extra- and intracellular nucleases and preferably have theability to recruit endogenous enzymes like RNAseH. In DNA-baseddiagnostics and molecular biology other properties are important suchas, e.g., the ability of oligonucleotides to act as efficient substratesfor a wide range of different enzymes evolved to act on natural nucleicacids, such as e.g. polymerases, kinases, ligases and phosphatases. Thefundamental property of oligonucleotides, however, which underlies alluses is their ability to recognise and hybridise sequence specificallyto complementary single stranded nucleic acids employing eitherWatson-Crick hydrogen bonding (A-T and G-C) or other hydrogen bondingschemes such as the Hoogsteen mode. The are two important terms affinityand specificity are commonly used to characterise the hybridisationproperties of a given oligonucleotide. Affinity is a measure of thebinding strength of the oligonucleotide to its complementary targetsequence (expressed as the thermostability (T_(m)) of the duplex). Eachnucleobase pair in the duplex adds to the thermostability and thusaffinity increases with increasing size (No. of nucleobases) of theoligonucleotide. Specificity is a measure of the ability of theoligonucleotide to discriminate between a fully complementary and amismatched target sequence. In other words, specificity is a measure ofthe loss of affinity associated with mismatched nucleobase pairs in thetarget. At constant oligonucleotide size the specificity increases withincreasing number of mismatches between the oligonucleotide and itstargets (i.e. the percentage of mismatches increases). Conversely,specificity decreases when the size of the oligonucleotide is increasedat a constant number of mismatches (i.e. the percentage of mismatchesdecreases). Stated another way, an increase in the affinity of anoligonucleotide occurs at the expense of specificity and vice-versa.

This property of oligonucleotides creates a number of problems for theirpractical use. In lengthy diagnostic procedures, for instance, theoligonucleotide needs to have both high affinity to secure adequatesensitivity of the test and high specificity to avoid false positiveresults. Likewise, an oligonucleotide used as antisense probes needs tohave both high affinity for its target mRNA to efficiently impair itstranslation and high specificity to avoid the unintentional blocking ofthe expression of other proteins. With enzymatic reactions, like, e.g.,PCR amplification, the affinity of the oligonucleotide primer must behigh enough for the primer/target duplex to be stable in the temperaturerange where the enzymes exhibits activity, and specificity needs to behigh enough to ensure that only the correct target sequence isamplified.

Given the shortcomings of natural oligonucleotides, new approaches forenhancing specificity and affinity would be highly useful for DNA-basedtherapeutics, diagnostics and for molecular biology techniques ingeneral.

Conformationally Restricted Nucleosides

It is known that oligonucleotides undergo a conformational transition inthe course of hybridising to a target sequence, from the relativelyrandom coil structure of the single stranded state to the orderedstructure of the duplex state.

A number of conformationally restricted oligonucleotides includingbicyclic and tricyclic nucleoside analogues (FIGS. 1A and 1B in whichB=nucleobase) have been synthesised, incorporated into oligonucleotideand oligonucleotide analogues and tested for their hybridisation andother properties.

Bicyclo[3.3.0] nucleosides (bcDNA) with an additionalC-3′,C-5′-ethano-bridge (A and B) have been synthesised with all fivenucleobases (G, A, T, C and U) whereas (C) has been synthesised onlywith T and A nucleobases (M. Tarköy, M. Bolli, B. Schweizer and C.Leumann, Helv. Chim. Acta, 1993, 76, 481; Tarköy and C. Leumann, Angew.Chem., Int. Ed. Engl., 1993, 32, 1432; M. Egli, P. Lubini, M. Dobler andC. Leumann, J. Am. Chem. Soc., 1993, 115, 5855; M. Tarkoy, M. Bolli andC. Leumann, Helv. Chim. Acta, 1994, 77, 716; M. Bolli and C. Leumann,Angew. Chem., Int. Ed. Engl., 1995, 34, 694; M. Bolli, P. Lubini and C.Leumann, Helv. Chim. Acta, 1995, 78, 2077; J. C. Litten, C. Epple and C.Leumann, Bioorg. Med. Chem. Lett., 1995, 5, 1231; J. C. Litten and C.Leumann, Helv. Chim. Acta, 1996, 79, 1129; M. Bolli, J. C. Litten, R.Schiiltz and C. Leumann, Chem. Biol., 1996, 3, 197; M. Bolli, H. U.Trafelet and C. Leumann, Nucleic Acids Res., 1996, 24, 4660). DNAoligonucleotides containing a few, or being entirely composed, of theseanalogues are in most cases able to form Watson-Crick bonded duplexeswith complementary DNA and RNA oligonucleotides. The thermostability ofthe resulting duplexes, however, is either distinctly lower (C),moderately lower (A) or comparable to (B) the stability of the naturalDNA and RNA counterparts. All bcDNA oligomers exhibited a pronouncedincrease in sensitivity to the ionic strength of the hybridisation mediacompared to the natural counterparts. The α-bicyclo-DNA (B) is morestable towards the 3′-exonuclease snake venom phosphordiesterase thanthe β-bicyclo-DNA (A) which is only moderately more stable thanunmodified oligonucleotides.

Bicarbocyclo[3.1.0]nucleosides with an additional C-1′, C-6′- or C-6′,C-4′-methano-bridge on a cyclopentane ring (D and E, respectively) havebeen synthesised with all five nucleobases (T, A, G, C and U). Only theT-analogues, however, have been incorporated into oligomers.Incorporation of one or ten monomers D in a mixed poly-pyrimidine DNAoligonucleotide resulted in a substantial decrease in the affinitytowards both DNA and RNA oligonucleotides compared to the unmodifiedreference oligonucleotide. The decrease was more pronounced with ssDNAthan with ssRNA. Incorporation of one monomer E in two differentpoly-pyrimidine DNA oligonucleotides induced modest increases in T_(m)'sof 0.8° C. and 2.1° C. for duplexes towards ssRNA compared withunmodified reference duplexes. When ten T-analogues were incorporatedinto a 15mer oligonucleotide containing exclusively phosphorothioateinternucleoside linkages, the T_(m) against the complementary RNAoligonucleotide was increased approximately 1.3° C. per modificationcompared to the same unmodified phosphorothioate sequence. Contrary tothe control sequence the oligonucleotide containing the bicyclicnucleoside E failed to mediate RNAseH cleavage. The hybridisationproperties of oligonucleotides containing the G, A, C and U-analogues ofE have not been reported. Also, the chemistry of this analogue does notlend itself to further intensive investigations on completely modifiedoligonucleotides (K.-H. Altmann, R. Kesselring, E. Francotte and G.Rihs, Tetrahedron Lett., 1994, 35, 2331; K.-H. Altmann, R. Imwinkelried,R. Kesselring and G. Rihs, Tetrahedron Lett., 1994, 35, 7625; V. E.Marquez, M. A. Siddiqui, A. Ezzitouni, P. Russ, J. Wang, R. W. Wagnerand M. D. Matteucci, J. Med. Chem., 1996, 39, 3739; A. Ezzitouni and V.E. Marquez, J. Chem. Soc., Perkin Trans. 1, 1997, 1073).

A bicyclo[3.3.0] nucleoside containing an additional C-2′,C-3′-dioxalanering has been synthesised as a dimer with an unmodified nucleoside wherethe additional ring is part of the internucleoside linkage replacing anatural phosphordiester linkage (F). This analogue was only synthesisedas either thymine-thymine or thymine-5-methylcytosine blocks. A 15-merpolypyrimidine sequence containing seven of these dimeric blocks andhaving alternating phosphordiester- and riboacetal-linkages, exhibited asubstantially decreased T_(m) against complementary ssRNA compared to acontrol sequence with exclusively natural phosphordiesterinternucleoside linkages (R. J. Jones, S. Swaminathan, J. F. Millagan,S. Wadwani, B. S. Froehler and M. Matteucci, J. Am. Chem. Soc., 1993,115, 9816).

The two dimers (G and H) with additional C-2′,C-3′-dioxane rings formingbicyclic[4.3.0]-systems in acetal-type internucleoside linkages havebeen synthesised as T-T dimers and incorporated once in the middle of12mer polypyrimidine oligonucleotides. Oligonucleotides containingeither G or H both formed significantly less stable duplexes withcomplementary ssRNA and ssDNA compared with the unmodified controloligonucleotide (J. Wang and M. D. Matteucci, Bioorg. Med. Chem. Lett.,1997, 7, 229).

Dimers containing a bicyclo[3.1.0]nucleoside with a C-2′,C-3′-methanobridge as part of amide- and sulfonamide-type (I and J) internucleosidelinkages have been synthesised and incorporated into oligonucleotides.Oligonucleotides containing one ore more of these analogues showed asignificant reduction in T_(m) compared to unmodified naturaloligonucleotide references (C. G. Yannopoulus, W. Q. Zhou, P. Nower, D.Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378).

A trimer with formacetal internucleoside linkages and a bicyclo[3.3.0]glucose-derived nucleoside analogue in the middle (K) has beensynthesised and connected to the 3′-end of an oligonucleotide. The T_(m)against complementary ssRNA was decreased by 4° C., compared to acontrol sequence, and by 1.5° C. compared to a sequence containing two2′,5′-formacetal linkages in the 3′-end (C. G. Yannopoulus, W. Q. Zhou,P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378).

Very recently oligomers composed of tricyclic nucleoside-analogues (L)have been reported to show increased duplex stability compared tonatural DNA (R. Steffens and C. Leumann (Poster SB-B4), Chimia, 1997,51, 436).

An attempt to make the bicyclic uridine nucleoside analogue Q planned tocontain an additional O-2′,C-4′-five-membered ring, starting from4′-C-hydroxymethyl nucleoside P, failed (K. D. Nielsen, Specialerapport(Odense University, Denmark), 1995).

Until now the pursuit of conformationally restricted nucleosides usefulin the formation of synthetic oligonucleotides with significantlyimproved hybridisation characteristics has met with little success. Inthe majority of cases, oligonucleotides containing these analogues formless stable duplexes with complementary nucleic acids compared to theunmodified oligonucleotides. In other cases, where moderate improvementin duplex stability is observed, this relates only to either a DNA or anRNA target, or it relates to fully but not partly modifiedoligonucleotides or vice versa. An appraisal of most of the reportedanalogues are further complicated by the lack of data on analogues withG, A and C nucleobases and lack of data indicating the specificity andmode of hybridisation. In many cases, synthesis of the reported monomeranalogues is very complex while in other cases the synthesis of fullymodified oligonucleotides is incompatible with the widely usedphosphoramidite chemistry standard.

BRIEF SUMMARY OF THE INVENTION

In view of the shortcomings of the previously known nucleosideanalogues, the present inventors have now provided novel nucleosideanalogues (LNAs) and oligonucleotides have included LNA nucleosideanalogues therein. The novel LNA nucleoside analogues have been providedwith all commonly used nucleobases thereby providing a full set ofnucleoside analogues for incorporation in oligonucleotides. As will beapparent from the following, the LNA nucleoside analogues and the LNAmodified oligonucleotide provides a wide range of improvements foroligonucleotides used in the fields of diagnostics and therapy.Furthermore, the LNA nucleoside analogues and the LNA modifiedoligonucleotide also provides completely new perspectives in nucleosideand oligonucleotide based diagnostics and therapy.

Thus, the present invention relates to oligomers comprising at least onenucleoside analogue (hereinafter termed “LNA”) of the general formula I

wherein X is selected from O, S, N(R^(N)*)—, —C(R⁶R⁶*)—, —O—C(R⁷R⁷*)—,—C(R⁶R⁶*)—O—, —S—C(R⁷R⁷*)—, —C(R⁶R⁶*)—S—, N(R^(N)*)—C(R⁷R⁷*)—,C(R⁶R⁶*)—N(R^(N)*)—, and —C(R⁶R⁶*)—C(R⁷R⁷*)—;B is selected from hydrogen, hydroxy, optionally substitutedC₁₋₄-alkoxy, optionally substituted C₁₋₄-alkyl, optionally substitutedC₁₋₄-acyloxy, nucleobases, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands;P designates the radical position for an internucleoside linkage to asucceeding monomer, or a 5′-terminal group, such internucleoside linkageor 5′-terminal group optionally including the substituent R⁵;one of the substituents R², R²*, R³, and R³* is a group P* whichdesignates an internucleoside linkage to a preceding monomer, or a3′-terminal group;one or two pairs of non-geminal substituents selected from the presentsubstituents of R¹*, R⁴*, R⁵, R⁵*, R⁶, R⁶*, R⁷, R⁷*, R^(N)*, and theones of R², R²*, R³, and R³* not designating P* each designates abiradical consisting of 1-8 groups/atoms selected from —C(R^(a)R^(b))—,—C(R^(a))═C(R^(a)), —C(R^(a))═N, O, —Si(R^(a))₂—, S—, SO₂—, —N(R^(a))—,and >C═Z,wherein Z is selected from —O—, —S—, and —N(R^(a))—, and R^(a) and R^(b)each is independently selected from hydrogen, optionally substitutedC₁₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionally substitutedC₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy, carboxy,C₁₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),and wherein two non-geminal or geminal substitutents selected fromR^(a), R^(b), and any of the substituents R¹*, R², R²*, R³, R³*, R⁴*,R⁵, R⁵*, R⁶ and R⁶*, R⁷, and R⁷* which are present and not involved inP, P* or the biradical(s) together may form an associated biradicalselected from biradicals of the same kind as defined before;said pair(s) of non-geminal substituents thereby forming a mono- orbicyclic entity together with (i) the atoms to which said non-geminalsubstituents are bound and (ii) any intervening atoms; andeach of the substituents R¹*, R², R²*, R³, R⁴*, R⁵, R⁵*, R⁶ and R⁶*, R⁷,and R⁷* which are present and not involved in P, P* or the biradical(s),is independently selected from hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy,carboxy, C₁₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and where two geminal substituents together maydesignate oxo, thioxo, imino, or optionally substituted methylene, ortogether may form a spiro biradical consisting of a 1-5 carbon atom(s)alkylene chain which is optionally interrupted and/or terminated by oneor more heteroatoms/groups selected from O, S, and —(NR^(N))— whereR^(N) is selected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N)*, when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl;and basic salts and acid addition salts thereof;with the proviso that,

-   (i) R³ and R⁵ do not together designate a biradical selected from    —CH₂—CH₂OCH₂—, when LNA is a bicyclic nucleoside analogue;-   (ii) R³, R⁵, and R⁵* do not together designate a triradical    CH₂CH(—)CH₂— when LNA is a tricyclic nucleoside analogue;-   (iii) R¹* and R⁶* do not together designate a biradical CH₂ when LNA    is a bicyclic nucleoside analogue; and-   (iv) R⁴* and R⁶* do not together designate a biradical CH₂ when LNA    is a bicyclic nucleoside analogue.

The present invention furthermore relates to nucleoside analogues(hereinafter LNAs) of the general formula II

wherein the substituent B is selected from nucleobases, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands;X is selected from —O—, —S—, —N(R^(N)*)—, and C(R⁶R⁶*)—;one of the substituents R², R²*, R³, and R³* is a group Q*;each of Q and Q* is independently selected from hydrogen, azido,halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—,Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted C₂₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl,optionally substituted C₂₋₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl;

-   (i) R²* and R⁴* together designate a biradical selected from —O—,    —(CR*R*)_(r+s+1)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,    (CR*R*)_(r)—S—(CR*R*)_(s)—, (CR*R*)_(r)—N(R*)—(CR*R*)_(s),    —O—(CR*R*)_(r+s)—O—, S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,    N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(s)—S—,    —N(R)(CR*R*)_(s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and    —S—(CR*R*)_(r+s)—(R*)—;-   (ii) R² and R³ together designate a biradical selected from O,    —(CR*R*)_(r+s)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,    (CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s);-   (iii) R²* and R³ together designate a biradical selected from O,    —(CR*R*)_(r+s)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,    (CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s);-   (iv) R³ and R⁴* together designate a biradical selected from    (CR*R*)_(r)—O—(CR*R*)_(s)—, (CR*R*)_(r)—S—(CR*R*)_(s)—, and    (CR*R*)_(r)—N(R*)—(CR*R*)_(s);-   (v) R³ and R⁵ together designate a biradical selected from    (CR*R*)_(r)—O—(CR*R*)_(s)—, (CR*R*)_(r)—S—(CR*R*)_(s)—, and    (CR*R*)_(r)—N(R*)—(CR*R*)_(s); or-   (vi) R¹* and R⁴* together designate a biradical selected from    (CR*R*)_(r)—O—(CR*R*)_(s), (CR*R*)_(r)—S—(CR*R*)_(s)—, and    (CR*R*)_(r)—N(R*)—(CR*R*)_(s);-   (vii) R¹* and R²* together designate a biradical selected from    (CR*R*)_(r)—O—(CR*R*)_(s), (CR*R*)_(r)—S—(CR*R*)_(s)—, and    (CR*R*)_(r)—N(R*)—(CR*R*)_(s);    -   wherein each R* is independently selected from hydrogen,        halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or        di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,        optionally substituted C₁₋₆-alkyl, DNA intercalators,        photochemically active groups, thermochemically active groups,        chelating groups, reporter groups, and ligands, and/or two        adjacent (non-geminal) R* may together designate a double bond,        and each of r and s is 0-3 with the proviso that the sum r+s is        1-4;        each of the substituents R¹*, R², R²*, R³, R⁴*, R⁵, and R⁵*,        which are not involved in Q, Q* or the biradical, is        independently selected from hydrogen, optionally substituted        C₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionally        substituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy,        C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,        C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,        arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,        heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino,        carbamoyl, mono- and di(C₁₋₆-alkyl)-amino-carbonyl,        amino-C₁₋₆-alkyl-aminocarbonyl, mono- and        di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,        C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,        C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,        halogen, DNA intercalators, photochemically active groups,        thermochemically active groups, chelating groups, reporter        groups, and ligands, where aryl and heteroaryl may be optionally        substituted, and where two geminal substituents together may        designate oxo, thioxo, imino, or optionally substituted        methylene, or together may form a spiro biradical consisting of        a 1-5 carbon atom(s) alkylene chain which is optionally        interrupted and/or terminated by one or more heteroatoms/groups        selected from O, S, and —(NR^(N))— where R^(N) is selected from        hydrogen and C₁₋₄-alkyl, and where two adjacent (non-geminal)        substituents may designate an additional bond resulting in a        double bond; and R^(N)*, when present and not involved in a        biradical, is selected from hydrogen and C₁₄-alkyl;        and basic salts and acid addition salts thereof;        with the first proviso that,-   (i) R³ and R⁵ do not together designate a biradical selected from    CH₂CH₂, OCH₂, and —O—Si(^(i)Pr)₂—O—Si(^(i)Pr)₂—O—;    and with the second proviso that any chemical group (including any    nucleobase), which is reactive under the conditions prevailing in    oligonucleotide synthesis, is optionally functional group protected.

The present invention also relates to the use of the nucleosideanalogues (LNAs) for the preparation of oligomers, and the use of theoligomers as well as the nucleoside analogues (LNAs) in diagnostics,molecular biology research, and in therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate known conformationally restrictednucleotides.

FIG. 2 illustrates nucleotide/nucleoside analogues of the invention.

FIG. 3 illustrates the performance of LNA modified oligonucleotides inthe sequence specific capture of PCR amplicons.

FIGS. 4A and 4B illustrate that LNA modified oligonucleotides are ableto capture its cognate PCR amplicon by strand invasion.

FIG. 5 illustrates that LNA modified oligonucleotides, immobilised on asolid surface, function efficiently in the sequence specific capture ofa PCR amplicon.

FIG. 6 illustrates that LNA modified oligonucleotides can act assubstrates for T4 polynucleotide kinase.

FIG. 7 illustrates that LNA modified oligonucleotides can function asprimers for nucleic acid polymerases.

FIG. 8 illustrates that LNA modified oligonucleotides can functions asprimers in target amplification processes.

FIG. 9 illustrates that LNA modified oligonucleotides carrying a 5′anthraquinone can be covalently immobilised on a solid support byirradiation and that the immobilised oligomer is efficient in thecapture of a complementary DNA oligo.

FIG. 10 illustrates that LNA-thymidine-5′-triphosphate (LNA-TTP) can actas a substrate for terminal deoxynucleotidyl transferase (TdT).

FIG. 11 illustrates hybridisation and detection on an array withdifferent LNA modified Cy3-labelled 8mers.

FIGS. 12 and 13 illustrate hybridisation and detection of end mismatcheson an array with LNA modified Cy3-labelled 8mers.

FIG. 14 illustrates blockade by LNA of [D-Ala2]deltorphin-inducedantinociception in the warm water tail flick test in conscious rats.

FIGS. 15A, 15B, and 15C illustrate Hybridization and detection of endmismatches on an array with AT and all LNA modified Cy3-labelled 8mers.

FIGS. 16 and 17 illustrate that LNA can be delivered to living humanMCF-7 breast cancer cells.

FIGS. 18 and 19 illustrate the use of [α³³P] ddNTP's andThermoSequenase™ DNA Polymerase to sequence DNA templates containing LNAT monomers.

FIGS. 20 and 21 illustrate that exonuclease free Klenow fragment DNApolymerase I can incorporate LNA Adenosine, Cytosine, Guanosine andUridine-5′-triphosphates into a DNA strand.

FIG. 22 illustrates the ability of terminal deoxynucleotidyl transferase(TdT) to tail LNA modified oligonucleotides.

FIGS. 23A and 23B illustrate that fully mixed LNA monomers can be usedto significantly increase the performance of immobilisedbiotinylated-DNA oligos in the sequence specific capture of PCRamplicons.

FIGS. 24 to 41 illustrates possible synthetic routes towards the LNAmonomers of the invention.

DETAILED DESCRIPTION OF THE INVENTION

When used herein, the term “LNA” (Locked Nucleoside Analogues) refers tothe bi- and tri-cyclic nucleoside analogues of the invention, eitherincorporated in the oligomer of the invention (general formula I) or asdiscrete chemical species (general formula II). The term “monomeric LNA”specifically refers to the latter case.

Oligomers and Nucleoside Analogues

As mentioned above, the present invention i.a. relates to noveloligomers (oligonucleotides) comprising one or more bi-, tri-, orpolycyclic nucleoside analogues (hereinafter termed “LNA”). It has beenfound that the incorporation of such LNAs in place of, or as asupplement to, e.g., known nucleosides confer interesting and highlyuseful properties to an oligonucleotide. Bi- and tricyclic, especiallybicyclic, LNAs seem especially interesting within the scope of thepresent invention.

Each of the possible LNAs incorporated in an oligomer (oligonucleotide)has the general formula I

wherein X is selected from —O— (the furanose motif), —S—, N(R^(N)*)—,—C(R⁶R⁶*)—, —O—C(R⁷R⁷*)—, —C(R⁶R⁶*)—O—, —S—C(R⁷R⁷*)—, C(R⁶R⁶*)—S—,N(R^(N)*)—C(R⁷R⁷*)—, C(R⁶R⁶*)—N(R^(N)*)—, and C(R⁶R⁶*)—C(R⁷R⁷*)—, whereR⁶, R⁶*, R⁷, R⁷*, and R^(N)* are as defined further below. Thus, theLNAs incorporated in the oligomer may comprise an either 5- or6-membered ring as an essential part of the bi-, tri-, or polycyclicstructure. It is believed that 5-membered rings (X═O—, —S—, N(R^(N)*)—,C(R⁶R⁶*)—) are especially interesting in that they are able to occupyessentially the same conformations (however locked by the introductionof one or more biradicals (see below)) as the native furanose ring of anaturally occurring nucleoside. Among the possible 5-membered rings, thesituations where X designates —O—, —S—, and N(R^(N)*)— seem especiallyinteresting, and the situation where X is O appears to be particularlyinteresting.

The substituent B may designate a group which, when the oligomer iscomplexing with DNA or RNA, is able to interact (e.g. by hydrogenbonding or covalent bonding or electronic interaction) with DNA or RNA,especially nucleobases of DNA or RNA. Alternatively, the substituent Bmay designate a group which acts as a label or a reporter, or thesubstituent B may designate a group (e.g. hydrogen) which is expected tohave little or no interactions with DNA or RNA. Thus, the substituent Bis preferably selected from hydrogen, hydroxy, optionally substitutedC₁₋₄-alkoxy, optionally substituted C₁₋₄-alkyl, optionally substitutedC₁₋₄-acyloxy, nucleobases, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands.

In the present context, the terms “nucleobase” covers naturallyoccurring nucleobases as well as non-naturally occurring nucleobases. Itshould be clear to the person skilled in the art that variousnucleobases which previously have been considered “non-naturallyoccurring” have subsequently been found in nature. Thus, “nucleobase”includes not only the known purine and pyrimidine heterocycles, but alsoheterocyclic analogues and tautomers thereof. Illustrative examples ofnucleobases are adenine, guanine, thymine, cytosine, uracil, purine,xanthine, diaminopurine, 8-oxo-N⁶-methyladenine, 7-deazaxanthine,7-deazaguanine, N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine,5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil,5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin,isocytosine, isoguanin, inosine and the “non-naturally occurring”nucleobases described in Benner et al., U.S. Pat. No. 5,432,272. Theterm “nucleobase” is intended to cover every and all of these examplesas well as analogues and tautomers thereof. Especially interestingnucleobases are adenine, guanine, thymine, cytosine, and uracil, whichare considered as the naturally occurring nucleobases in relation totherapeutic and diagnostic application in humans.

When used herein, the term “DNA intercalator” means a group which canintercalate into a DNA or RNA helix, duplex or triplex. Examples offunctional parts of DNA intercalators are acridines, anthracene,quinones such as anthraquinone, indole, quinoline, isoquinoline,dihydroquinones, anthracyclines, tetracyclines, methylene blue,anthracyclinone, psoralens, coumarins, ethidium-halides, dynemicin,metal complexes such as 1,10-phenanthroline-copper,tris(4,7-diphenyl-1,10-phenanthroline)ruthenium-cobalt-enediynes such ascalcheamicin, porphyrins, distamycin, netropcin, viologen, daunomycin.Especially interesting examples are acridines, quinones such asanthraquinone, methylene blue, psoralens, coumarins, andethidium-halides.

In the present context, the term “photochemically active groups” coverscompounds which are able to undergo chemical reactions upon irradiationwith light. Illustrative examples of functional groups hereof arequinones, especially 6-methyl-1,4-naphtoquinone, anthraquinone,naphtoquinone, and 1,4-dimethyl-anthraquinone, diazirines, aromaticazides, benzophenones, psoralens, diazo compounds, and diazirinocompounds.

In the present context “thermochemically reactive group” is defined as afunctional group which is able to undergo thermochemically-inducedcovalent bond formation with other groups. Illustrative examples offunctional parts thermochemically reactive groups are carboxylic acids,carboxylic acid esters such as activated esters, carboxylic acid halidessuch as acid fluorides, acid chlorides, acid bromide, and acid iodides,carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids,sulfonic acid esters, sulfonic acid halides, semicarbazides,thiosemicarbazides, aldehydes, ketones, primary alkohols, secondaryalkohols, tertiary alkohols, phenols, alkyl halides, thiols,disulphides, primary amines, secondary amines, tertiary amines,hydrazines, epoxides, maleimides, and boronic acid derivatives.

In the present context, the term “chelating group” means a molecule thatcontains more than one binding site and frequently binds to anothermolecule, atom or ion through more than one binding site at the sametime. Examples of functional parts of chelating groups are iminodiaceticacid, nitrilotriacetic acid, ethylenediamine tetraacetic acid (EDTA),aminophosphonic acid, etc.

In the present context, the term “reporter group” means a group which isdetectable either by itself or as a part of an detection series.Examples of functional parts of reporter groups are biotin, digoxigenin,fluorescent groups (groups which are able to absorb electromagneticradiation, e.g. light or X-rays, of a certain wavelength, and whichsubsequently reemits the energy absorbed as radiation of longerwavelength; illustrative examples are dansyl(5-dimethylamino)-1-naphthalenesulfonyl), DOXYL(N-oxyl-4,4-dimethyloxazolidine), PROXYL(N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO(N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines,coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems,Inc.), erytrosine, coumaric acid, umbelliferone, texas red, rhodamine,tetramethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene,fluorescein, Europium, Ruthenium, Samarium, and other rare earthmetals), radioisotopic labels, chemiluminescence labels (labels that aredetectable via the emission of light during a chemical reaction), spinlabels (a free radical (e.g. substituted organic nitroxides) or otherparamagnetic probes (e.g. Cu²⁺, Mg²⁺) bound to a biological moleculebeing detectable by the use of electron spin resonance spectroscopy),enzymes (such as peroxidases, alkaline phosphatases, β-galactosidases,and glycose oxidases), antigens, antibodies, haptens (groups which areable to combine with an antibody, but which cannot initiate an immuneresponse by itself, such as peptides and steroid hormones), carriersystems for cell membrane penetration such as: fatty acid residues,steroid moieties (cholesteryl), vitamin A, vitamin D, vitamin E, folicacid peptides for specific receptors, groups for mediating endocytose,epidermal growth factor (EGF), bradykinin, and platelet derived growthfactor (PDGF). Especially interesting examples are biotin, fluorescein,Texas Red, rhodamine, dinitrophenyl, digoxigenin, Ruthenium, Europium,Cy5, Cy3, etc.

In the present context “ligand” means something which binds. Ligands cancomprise functional groups such as: aromatic groups (such as benzene,pyridine, naphtalene, anthracene, and phenanthrene), heteroaromaticgroups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane,and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylicacid halides, carboxylic acid azides, carboxylic acid hydrazides,sulfonic acids, sulfonic acid esters, sulfonic acid halides,semicarbazides, thiosemicarbazides, aldehydes, ketones, primaryalcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides,thiols, disulphides, primary amines, secondary amines, tertiary amines,hydrazines, epoxides, maleimides, C₁-C₂₀ alkyl groups optionallyinterrupted or terminated with one or more heteroatoms such as oxygenatoms, nitrogen atoms, and/or sulphur atoms, optionally containingaromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such aspolyethylene glycol, oligo/polyamides such as poly-β-alanine,polyglycine, polylysine, peptides, oligo/polysaccharides,oligo/polyphosphates, toxins, antibiotics, cell poisons, and steroids,and also “affinity ligands”, i.e. functional groups or biomolecules thathave a specific affinity for sites on particular proteins, antibodies,poly- and oligosaccharides, and other biomolecules.

It will be clear for the person skilled in the art that theabove-mentioned specific examples under DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands correspond to the“active/functional” part of the groups in question. For the personskilled in the art it is furthermore clear that DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands are typically represented in theform M-K- where M is the “active/functional” part of the group inquestion and where K is a spacer through which the “active/functional”part is attached to the 5- or 6-membered ring. Thus, it should beunderstood that the group B, in the case where B is selected from DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, has the formMK-, where M is the “active/functional” part of the DNA intercalator,photochemically active group, thermochemically active group, chelatinggroup, reporter group, and ligand, respectively, and where K is anoptional spacer comprising 1-50 atoms, preferably 1-30 atoms, inparticular 1-15 atoms, between the 5- or 6-membered ring and the“active/functional” part.

In the present context, the term “spacer” means a thermochemically andphotochemically non-active distance-making group and is used to join twoor more different moieties of the types defined above. Spacers areselected on the basis of a variety of characteristics including theirhydrophobicity, hydrophilicity, molecular flexibility and length (e.g.see Hermanson et. al., “Immobilized Affinity Ligand Techniques”,Academic Press, San Diego, Calif. (1992), p. 137-ff). Generally, thelength of the spacers are less than or about 400 Å, in some applicationspreferably less than 100 Å. The spacer, thus, comprises a chain ofcarbon atoms optionally interrupted or terminated with one or moreheteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulphur atoms.Thus, the spacer K may comprise one or more amide, ester, amino, ether,and/or thioether functionalities, and optionally aromatic ormono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethyleneglycol, oligo/polyamides such as poly-β-alanine, polyglycine,polylysine, and peptides in general, oligosaccharides,oligo/polyphosphates. Moreover the spacer may consist of combined unitsthereof. The length of the spacer may vary, taking into considerationthe desired or necessary positioning and spatial orientation of the“active/functional” part of the group in question in relation to the 5-or 6-membered ring. In particularly interesting embodiments, the spacerincludes a chemically cleavable group. Examples of such chemicallycleavable groups include disulphide groups cleavable under reductiveconditions, peptide fragments cleavable by peptidases, etc.

In one embodiment of the present invention, K designates a single bondso that the “active/functional” part of the group in question isattached directly to the 5- or 6-membered ring.

In a preferred embodiment, the substituent B in the general formulae Iand II is preferably selected from nucleobases, in particular fromadenine, guanine, thymine, cytosine and urasil.

In the oligomers of the present invention (formula I), P designates theradical position for an internucleoside linkage to a succeeding monomer,or a 5′-terminal group. The first possibility applies when the LNA inquestion is not the 5′-terminal “monomer”, whereas the latterpossibility applies when the LNA in question is the 5′-terminal“monomer”. It should be understood (which also will be clear from thedefinition of internucleoside linkage and 5′-terminal group furtherbelow) that such an internucleoside linkage or 5′-terminal group mayinclude the substituent R⁵ (or equally applicable: the substituent R⁵*)thereby forming a double bond to the group P. (5′-Terminal refers to theposition corresponding to the 5′ carbon atom of a ribose moiety in anucleoside.)

On the other hand, an internucleoside linkage to a preceding monomer ora 3′-terminal group (P*) may originate from the positions defined by oneof the substituents R², R²*, R³, and R³*, preferably from the positionsdefined by one of the substituents R³ and R³*. Analogously, the firstpossibility applies where the LNA in question is not the 3′-terminal“monomer”, whereas the latter possibility applies when the LNA inquestion is the 3′-terminal “monomer”. (3′-Terminal refers to theposition corresponding to the 3′ carbon atom of a ribose moiety in anucleoside.)

In the present context, the term “monomer” relates to naturallyoccurring nucleosides, non-naturally occurring nucleosides, PNAs, etc.as well as LNAs. Thus, the term “succeeding monomer” relates to theneighbouring monomer in the 5′-terminal direction and the “precedingmonomer” relates to the neighbouring monomer in the 3′-terminaldirection. Such succeeding and preceding monomers, seen from theposition of an LNA monomer, may be naturally occurring nucleosides ornon-naturally occurring nucleosides, or even further LNA monomers.

Consequently, in the present context (as can be derived from thedefinitions above), the term “oligomer” means an oligonucleotidemodified by the incorporation of one or more LNA(s).

The crucial part of the present invention is the presence of one or morerings fused to the 5- or 6-membered ring illustrated with the generalformula I. Thus, one or two pairs of non-geminal substituents selectedfrom the present substituents of R¹*, R⁴*, R⁵, R⁵*, R⁶, R⁶*, R⁷, R⁷*,R^(N)*, and the ones of R², R²*, R³, and R³* not designating P* eachdesignates a biradical consisting of 1-8 groups/atoms, preferably 1-4groups/atoms, independently selected from C(R^(a)R^(b))—,—C(R^(a))═C(R^(a)), —C(R^(a))═N, O, Si(R^(a))₂—, S—, —SO₂—, —N(R^(a))—,and >C═Z. (The term “present” indicates that the existence of some ofthe substituents, i.e. R⁶, R⁶*, R⁷, R⁷*, R^(N)*, is dependent on whetherX includes such substituents.)

In the groups constituting the biradical(s), Z is selected from O—, —S—,and —N(R^(a))—, and R^(a) and R^(b) each is independently selected fromhydrogen, optionally substituted C₁₋₁₂-alkyl, optionally substitutedC₂₋₄₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl, hydroxy,C₁₋₁₂-alkoxy, C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl,mono- and di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands (where the latter groups may include a spacer as defined for thesubstituent B), where aryl and heteroaryl may be optionally substituted.Moreover, two geminal substituents R^(a) and R^(b) together maydesignate optionally substituted methylene (═CH₂ optionally substitutedone or two times with substituents as defined as optional substituentsfor aryl), and two non-geminal or geminal substituents selected fromR^(a), R^(b), and any of the substituents R¹*, R², R²*, R³, R³*, R⁴*,R⁵, R⁵*, R⁶ and R⁶*, R⁷, and R⁷* which are present and not involved inP, P* or the biradical(s) may together form an associated biradicalselected from biradicals of the same kind as defined before. It will beclear that each of the pair(s) of non-geminal substituents thereby formsa mono- or bicyclic entity together with (i) the atoms to which thenon-geminal substituents are bound and (ii) any intervening atoms.

It is believed that biradicals which are bound to the ring atoms of the5- or 6-membered rings are preferred in that inclusion of thesubstituents R⁵ and R⁵* may cause an undesired sterical interaction withinternucleoside linkage. Thus, it is preferred that the one or two pairsof non-geminal substituents, which are constituting one or twobiradical(s), respectively, are selected from the present substituentsof R¹*, R⁴*, R⁶, R⁶*, R⁷, R⁷*, R^(N)*, and the ones of R², R²*, R³, andR³* not designating P.

Preferably, the LNAs incorporated in the oligomers comprise only onebiradical constituted by a pair of (two) non-geminal substituents. Inparticular, it is preferred that R³* designates P* and that thebiradical is formed between R²* and R⁴* or R² and R³.

This being said, it should be understood (especially with dueconsideration of the known bi- and tricyclic nucleoside analogues—see“Background of the Invention”) that the present invention does notrelate to oligomers comprising the following bi- or tricyclicnucleosides analogues:

-   (i) R² and R³ together designate a biradical selected from OCH₂CH₂    and O—CH₂CH₂CH₂ when LNA is a bicyclic nucleoside analogue;-   (ii) R³ and R⁵ together designate a biradical selected from CH₂CH₂,    OCH₂, when LNA is a bicyclic nucleoside analogue;-   (iii) R³, R⁵, and R⁵* together designate a triradical CH₂CH(−)CH₂—    when LNA is a tricyclic nucleoside analogue;-   (iv) R¹* and R⁶* together designate a biradical CH₂ when LNA is a    bicyclic nucleoside analogue; or-   (v) R⁴* and R⁶* together designate a biradical CH₂ when LNA is a    bicyclic nucleoside analogue;    except where such bi- or tricyclic nucleoside analogues are combined    with one or more of the novel LNAs defined herein.

In the present context, i.e. in the present description and claims, theorientation of the biradicals are so that the left-hand side representsthe substituent with the lowest number and the right-hand siderepresents the substituent with the highest number, thus, when R³ and R⁵together designate a biradical “O—CH₂—”, it is understood that theoxygen atom represents R³, thus the oxygen atom is e.g. attached to theposition of R³, and the methylene group represents R⁵.

Considering the numerous interesting possibilities for the structure ofthe biradical(s) in LNA(s) incorporated in oligomers according to theinvention, it is believed that the biradical(s) constituted by pair(s)of non-geminal substituents preferably is/are selected from—(CR*R*)_(r)—Y—(CR*R*)_(s)—, (CR*R*)_(r)—Y—(CR*R*)_(s)—Y—,—Y—(CR*R*)_(r), —Y—, —Y—(CR*R*)_(r)—Y—(CR*R*)_(s)—, —(CR*R*)_(r+s)—, Y—,—Y—Y—, wherein each Y is independently selected from O, S, —Si(R*)₂—,—N(R*), >C═O, —C(═O)—N(R*)—, and N(R*)—C(═O), each R* is independentlyselected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto,amino, mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₋₆-alkoxy,optionally substituted C₁₋₆-alkyl, DNA intercalators, photochemicallyactive groups, thermochemically active groups, chelating groups,reporter groups, and ligands, and/or two adjacent (non-geminal) R* maytogether designate a double bond; and each of r and s is 0-4 with theproviso that the sum r+s is 15. Particularly interesting situations arethose wherein each biradical is independently selected from Y,(CR*R*)_(r+s), (CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R)_(r+s)—Y—,wherein and each of r and s is 0-3 with the proviso that the sum r+s is1-4.

Considering the positioning of the biradical in the LNA(s), it isbelieved (based on the preliminary findings (see the examples)) that thefollowing situations are especially interesting, namely where: R²* andR⁴* together designate a biradical selected from Y, —(CR*R*)_(r+s+1)—,(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; R² and R³ togetherdesignate a biradical selected from Y, —(CR*R*)_(r+s)—,(CR*R*)_(r)—Y—(CR*R*)_(s), and —Y—(CR*R*)_(r+s)—Y—; R²* and R³ togetherdesignate a biradical selected from Y, —(CR*R*)_(r+s)—,(CR*R*)_(r)—Y—(CR*R*)_(s), and —Y—(CR*R*)_(r+s)—Y; R³ and R⁴* togetherdesignate a biradical selected from Y, —(CR*R*)_(r+s)—,(CR*R)_(r)—Y—(CR*R*)_(s), and —Y—(CR*R*)_(r+s)—Y—; R³ and R⁵ togetherdesignate a biradical selected from Y′, —(CR*R*)_(r+s+1)—,(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; R¹* and R⁴*together designate a biradical selected from Y′, —(CR*R*)_(r+s+1)—,(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—NR*—; or where R¹* andR²* together designate a biradical selected from Y, —(CR*R*)_(r+s)—,(CR*R*)_(r)—Y—(CR*R*)_(s)—, and —Y—(CR*R*)_(r+s)—Y—; wherein each of rand s is 0-3 with the proviso that the sum r+s is 1-4, Y is as definedabove, and where Y′ is selected from —NR*—C(═O)— and —C(═O)—NR*.

Particularly interesting oligomers are those wherein one of thefollowing criteria applies for at least one LNA in an oligomer: R²* andR⁴* together designate a biradical selected from —O—, —S—, N(R*)—,—(CR*R*)_(r+s+1)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, (CR*R*)_(r)—N(R*)—(CR*R*)_(s),—O—(CR*R*)_(r+s)—O—, S—(CR*R*)_(r+s)—O—, —O—(CR*R)_(r+s)—S—,N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, S—(CR*R*)_(r+s)—S—,—N(R*)(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CRR*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; R² and R³ together designate a biradicalselected from O, —(CR*R*)_(r+s)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); R²* andR³ together designate a biradical selected from O, (CR*R*)_(r+s)—,(CR*R*)_(r)—O—(CR*R*)_(r)—O—(CR*R*)_(r)—S—(CR*R*)_(s)—, and(CR*R*)_(r)—N(R*)—(CR*R*)_(s); R³ and R⁴* together designate a biradicalselected from (CR*R*)_(r)—O—(CR*R*)_(s)—, (CR*R*)_(r)—S—(CR*R*)_(s)—,and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); R³ and R⁵ together designate abiradical selected from (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); R¹* andR⁴* together designate a biradical selected from(CR*R*)_(r)—O—(CR*R*)_(s)—, (CR*R*)_(r)—S—(CR*R*)_(s)—, and(CR*R*)_(r)—N(R*)—(CR*R*)_(s); or R¹* and R²* together designate abiradical selected from (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); whereineach of r and s is 0-3 with the proviso that the sum r+s is 1-4, andwhere R^(H) designates hydrogen or C₁₋₄-alkyl.

It is furthermore preferred that one R* is selected from hydrogen,hydroxy, optionally substituted C₁₋₆-alkoxy, optionally substitutedC₁₋₆-alkyl, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands, and any remaining substituents R* are hydrogen.

In one preferred embodiment, one group R* in the biradical of at leastone LNA is selected from DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands (where the latter groups may include a spacer asdefined for the substituent B).

With respect to the substituents R¹*, R², R²*, R³, R⁴*, R⁵, R⁵*, R⁶ andR⁶*, R⁷, and R⁷*, which are present and not involved in P, P* or thebiradical(s), these are independently selected from hydrogen, optionallysubstituted C₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl,optionally substituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands (where the latter groups mayinclude a spacer as defined for the substituent B), where aryl andheteroaryl may be optionally substituted, and where two geminalsubstituents together may designate oxo, thioxo, imino, or optionallysubstituted methylene, or together may form a spiro biradical consistingof a 1-5 carbon atom(s) alkylene chain which is optionally interruptedand/or terminated by one or more heteroatoms/groups selected from O, S,and —(NR^(N))— where R^(N) is selected from hydrogen and C₁₋₄-alkyl, andwhere two adjacent (non-geminal) substituents may designate anadditional bond resulting in a double bond; and R^(N)*, when present andnot involved in a biradical, is selected from hydrogen and C₁₋₄-alkyl.

Preferably, each of the substituents R¹*, R², R²*, R³, R³*, R⁴*, R⁵,R⁵*, R⁶, R⁶*, R⁷, and R⁷* of the LNA(s), which are present and notinvolved in P, P* or the biradical(s), is independently selected fromhydrogen, optionally substituted C₁₋₆-alkyl, optionally substitutedC₂₋₆-alkenyl, hydroxy, C₁₋₆-alkoxy, C₂₋₆-alkenyloxy, carboxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl, amino, mono- anddi(C₁₋₆-alkyl)amino, carbamoyl, mono- and di(C₁₋₆-alkyl)-amino-carbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, azido, C₁₋₆-alkanoyloxy, sulphono,sulphanyl, C₁₋₆-alkylthio, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and halogen, where two geminal substituentstogether may designate oxo, and where R^(N)*, when present and notinvolved in a biradical, is selected from hydrogen and C₁₋₄-alkyl.

In a preferred embodiment of the present invention, X is selected fromO, S, and NR^(N)*—, in particular —O—, and each of the substituents R¹*,R², R²*, R³, R³*, R⁴*, R⁵, R⁵*, R⁶, R⁶*, R⁷, and R⁷* of the LNA(s),which are present and not involved in P, P* or the biradical(s),designate hydrogen.

In an even more preferred embodiment of the present invention, R²* andR⁴* of an LNA incorporated into an oligomer together designate abiradical. Preferably, X is O, R² selected from hydrogen, hydroxy, andoptionally substituted C₁₋₆-alkoxy, and R¹*, R³, R⁵, and R⁵* designatehydrogen, and, more specifically, the biradical is selected from —O—,(CH₂)₀₋₁—O(CH₂)₁₋₃—, (CH₂)₀₋₁—S(CH₂)₁₋₃—, (CH₂)₀₋₁—N(R^(N))(CH₂)₁₋₃—,and (CH₂)₂₋₄—, in particular from —O—CH₂—, —S—CH₂—, and —NR^(H)—CH₂—.Generally, with due regard to the results obtained so far, it ispreferred that the biradical constituting R²* and R⁴* forms a two carbonatom bridge, i.e. the biradical forms a five membered ring with thefuranose ring (X═O).

In another embodiment of the present invention, R² and R³ of an LNAincorporated into an oligomer together designate a biradical.Preferably, X is O, R²* is selected from hydrogen, hydroxy, andoptionally substituted C₁₋₆-alkoxy, and R¹*, R⁴*, R⁵, and R⁵* designatehydrogen, and, more specifically, the biradical is selected from(CH₂)₀₋₁—O(CH₂)₁₋₃—, (CH₂)₀₋₁—S(CH₂)₁₋₃—, (CH₂)₀₋₁—N(R^(H))(CH₂)₁₋₃— and(CH₂)₁₋₄—, in particular from OCH₂—, —SCH₂—, N(R^(H))CH₂—. In the lattercase, the amino and thio variants appears to be particularlyinteresting.

In a further embodiment of the present invention, R²* and R³ of an LNAincorporated into an oligomer together designate a biradical.Preferably, X is O, R² is selected from hydrogen, hydroxy, andoptionally substituted C₁₋₆-alkoxy, and R¹*, R⁴*, R⁵, and R⁵* designatehydrogen, and, more specifically, the biradical is selected from(CH₂)₀₋₁—O(CH₂)₁₋₃— and (CH₂)₂₋₄—.

In a further embodiment of the present invention, R³ and R⁴* of an LNAincorporated into an oligomer together designate a biradical.Preferably, X is O, R²* selected from hydrogen, hydroxy, and optionallysubstituted C₁₋₆-alkoxy, and R¹*, R², R⁵, and R⁵* designate hydrogen,and, more specifically, the biradical is (CH₂)₀₋₂—O(CH₂)₀₋₂—.

In a further embodiment of the present invention, R³ and R⁵* of an LNAincorporated into an oligomer together designate a biradical.Preferably, X is O, R²* selected from hydrogen, hydroxy, and optionallysubstituted C₁₋₆-alkoxy, and R¹*, R², R⁴, and R⁵ designate hydrogen,and, more specifically, the biradical is selected from O—(CHR*)₂₋₃— and(CHR*)₁₋₃—O—(CHR*)₀₋₃—.

In a further embodiment of the present invention, R¹* and R⁴* of an LNAincorporated into an oligomer together designate a biradical.Preferably, X is O, R²* selected from hydrogen, hydroxy, and optionallysubstituted C₁₋₆-alkoxy, and R², R³, R⁵, and R⁵* designate hydrogen,and, more specifically, the biradical is (CH₂)₀₋₂—O(CH₂)₀₋₂—.

In these embodiments, it is furthermore preferred that at least one LNAincorporated in an oligomer includes a nucleobase (substituent B)selected from adenine and guanine. In particular, it is preferred thatan oligomer have LNA incorporated therein both include at least onenucleobase selected from thymine, urasil and cytosine and at least onenucleobase selected from adenine and guanine. For LNA monomers, it isespecially preferred that the nucleobase is selected from adenine andguanine.

For these interesting embodiments, it is also preferred that the LNA(s)has/have the general formula Ia (see below).

Within a variant of these interesting embodiments, all monomers of aoligonucleotide are LNA monomers.

As it will be evident from the general formula I (LNA(s) in an oligomer)(and the general formula II (monomeric LNA)—see below) and thedefinitions associated therewith, there may be one or several asymmetriccarbon atoms present in the oligomers (and monomeric LNAs) depending onthe nature of the substituents and possible biradicals, cf. below. Theoligomers prepared according to the method of the invention, as well asthe oligomers per se, are intended to include all stereoisomers arisingfrom the presence of any and all isomers of the individual monomerfragments as well as mixtures thereof, including racemic mixtures. Whenconsidering the 5- or 6-membered ring, it is, however, believed thatcertain stereochemical configurations will be especially interesting,e.g. the following

where the wavy lines represent the possibility of both diastereomersarising from the interchange of the two substituents in question.

An especially interesting stereoisomeric representation is the casewhere the LNA(s) has/have the following formula Ia

Also interesting as a separate aspect of the present invention is thevariant of formula Ia where B is in the “α-configuration”.

In these cases, as well as generally, R³* preferably designates P*.

The oligomers according to the invention typically comprise 1-10000LNA(s) of the general formula I (or of the more detailed general formulaIa) and 0-10000 nucleosides selected from naturally occurringnucleosides and nucleoside analogues. The sum of the number ofnucleosides and the number of LNA(s) is at least 2, preferably at least3, in particular at least 5, especially at least 7, such as in the rangeof 2-15000, preferably in the range of 2-100, such as 3-100, inparticular in the range of 2-50, such as 3-50 or 5-50 or 7-50.

Preferably at least one LNA comprises a nucleobase as the substituent B.

In the present context, the term “nucleoside” means a glycoside of aheterocyclic base. The term “nucleoside” is used broadly as to includenon-naturally occurring nucleosides, naturally occurring nucleosides aswell as other nucleoside analogues. Illustrative examples of nucleosidesare ribonucleosides comprising a ribose moiety as well asdeoxyribonuclesides comprising a deoxyribose moiety. With respect to thebases of such nucleosides, it should be understood that this may be anyof the naturally occurring bases, e.g. adenine, guanine, cytosine,thymine, and uracil, as well as any modified variants thereof or anypossible unnatural bases.

When considering the definitions and the known nucleosides (naturallyoccurring and non-naturally occurring) and nucleoside analogues(including known bi- and tricyclic analogues), it is clear that anoligomer may comprise one or more LNA(s) (which may be identical ordifferent both with respect to the selection of substituent and withrespect to selection of biradical) and one or more nucleosides and/ornucleoside analogues. In the present context “oligonucleotide” means asuccessive chain of nucleosides connected via internucleoside linkages,however, it should be understood that a nucleobase in one or morenucleotide units (monomers) in an oligomer (oligonucleotide) may havebeen modified with a substituent B as defined above.

The oligomers may be linear, branched or cyclic. In the case of abranched oligomer, the branching points may be located in a nucleoside,in an internucleoside linkage or, in an intriguing embodiment, in anLNA. It is believed that in the latter case, the substituents R², R²*,R³, and R³* may designate two groups P* each designating aninternucleoside linkage to a preceding monomer, in particular, one of R²and R²* designate P* and one or R³ and R³* designate a further P.

As mentioned above, the LNA(s) of an oligomer are connected with othermonomers via an internucleoside linkage. In the present context, theterm “internucleoside linkage” means a linkage consisting of 2 to 4,preferably 3, groups/atoms selected from —CH₂—, O, S—, NR^(H)—, >C═O,>C═NR^(H), >C═S, Si(R″)₂—, —SO—, —S(O)₂—, P(O)₂—, PO(BH₃)—, P(O,S)—,P(S)₂—, PO(R″), PO(OCH₃), and —PO(NHR^(H))—, where R^(H) is selectedform hydrogen and C₁₋₄-alkyl, and R″ is selected from C₁₋₆-alkyl andphenyl. Illustrative examples of such internucleoside linkages areCH₂—CH₂—CH₂—, —CH₂—CO—CH₂—, —CH₂—CHOH—CH₂—, OCH₂—O, OCH₂—CH₂—, O—CH₂—CH═(including R⁵ when used as a linkage to a succeeding monomer),CH₂—CH₂—O, NR^(H)CH₂—CH₂—, CH₂—CH₂—NR^(H), —CH₂—NR^(H)—CH₂,OCH₂—CH₂—NR^(H), NR^(H)COO, NR^(H)CONR^(H), NR^(H)CSNR^(H),NR^(H)C(═NR^(H))NR^(H), —NR^(H)—COCH₂NR^(H), —O—CO—O—, —O—CO—CH₂—O—,—O—CH₂—CO—O—, CH₂—CO—NR^(H)—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—,—OCH₂—CO—NR^(H), —OCH₂—CH₂—NR^(H)—, —CH═N—O—, CH₂—NR^(H)—O—, —CH₂—O—N═(including R⁵ when used as a linkage to a succeeding monomer),—CH₂—O—NR^(H)—, CONR^(H)—CH₂, CH₂—NR^(H)—O—, CH₂—NR^(H)—CO—,ONR^(H)—CH₂—, ONR^(H)—, O—CH₂—S—, SCH₂—O—, CH₂—CH₂—S, O—CH₂—CH₂—S—,—S—CH₂—CH═ (including R⁵ when used as a linkage to a succeedingmonomer), —S—CH₂—CH₂—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—S—CH₂—, CH₂—S,OCH₂—, —CH₂—SO₂CH₂—, OSOO, —O—S(O)₂—O—, —O—S(O)₂—CH₂, O—S(O)₂—NR^(H),NR^(H)S(O)₂CH₂, OS(O)₂CH₂—, —O—P(O)₂—O, OP(O,S)—O—, —O—P(S)₂O—,—S—P(O)₂—O—, SP(O,S)—O—, —S—P(S)₂O—, —O—P(O)₂—S—, OP(O,S)—S—,—O—P(S)₂S—, —S—P(O)₂—S—, SP(O,S)—S—, —S—P(S)₂S—, OPO(R″)O—,—O—PO(OCH₃)—O—, —O—PO(OCH₂CH₃)—O—, —O—PO(OCH₂CH₂S—R)—O—, OPO(BH₃)—O—,O—PO(NHR^(N))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—,—CH₂—P(O)₂—O, —O—P(O)₂—CH₂, and —O—Si(R″)₂—O—; among whichCH₂—CO—NR^(H)—, CH₂—NR^(H)—O—, —S—CH₂—O—, —O—P(O)₂—O—, OP(O,S)—O—,—O—P(S)₂O—, —NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, OPO(R″)O—, OPO(CH₃)O,and O—PO(NHR^(N))—O—, where R^(H) is selected form hydrogen andC₁₋₄-alkyl, and R″ is selected from C₁₋₆-alkyl and phenyl, areespecially preferred. Further illustrative examples are given inMesmaeker et. al., Current Opinion in Structural Biology 1995, 5,343-355. The left-hand side of the internucleoside linkage is bound tothe 5- or 6-membered ring as substituent P*, whereas the right-hand sideis bound to the 5′-position of a preceding monomer.

It is also clear from the above that the group P may also designate a5′-terminal group in the case where the LNA in question is the5′-terminal monomer. Examples of such 5′-terminal groups are hydrogen,hydroxy, optionally substituted C₁₋₆-alkyl, optionally substitutedC₁₋₆-alkoxy, optionally substituted C₁₋₆-alkylcarbonyloxy, optionallysubstituted aryloxy, monophosphate, diphosphate, triphosphate, and—W-A′, wherein W is selected from O, S, and N(R^(H))— where R^(H) isselected from hydrogen and C₁₋₆-alkyl, and where A′ is selected from DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands (where the lattergroups may include a spacer as defined for the substituent B).

In the present description and claims, the terms “monophosphate”,“diphosphate”, and “triphosphate” mean groups of the formula:—O—P(O)₂—O⁻, —O—P(O)₂—O—P(O)₂—O⁻, and —O—P(O)₂—O—P(O)₂—O—P(O)₂—O⁻,respectively.

In a particularly interesting embodiment, the group P designates a5′-terminal groups selected from monophosphate, diphosphate andtriphosphate. Especially the triphosphate variant is interesting as asubstrate

Analogously, the group P* may designate a 3′-terminal group in the casewhere the LNA in question is the 3′-terminal monomer. Examples of such3′-terminal groups are hydrogen, hydroxy, optionally substitutedC₁₋₆-alkoxy, optionally substituted C₁₋₆-alkylcarbonyloxy, optionallysubstituted aryloxy, and —W-A′, wherein W is selected from O, S, andN(R^(H))— where R^(H) is selected from hydrogen and C₁₋₆-alkyl, andwhere A′ is selected from DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands (where the latter groups may include a spacer asdefined for the substituent B).

In a preferred embodiment of the present invention, the oligomer has thefollowing formula V:G-[Nu-L]_(n(0))-{[LNA-L]_(m(q))-[Nu-L]_(n(q))}_(q)-G*  Vwhereinq is 1-50;each of n(0), . . . , n(q) is independently 0-10000;each of m(1), . . . , m(q) is independently 1-10000;with the proviso that the sum of n(0), . . . , n(q) and m(1), . . . ,m(q) is 2-15000;G designates a 5′-terminal group;each Nu independently designates a nucleoside selected from naturallyoccurring nucleosides and nucleoside analogues;each LNA independently designates a nucleoside analogue;each L independently designates an internucleoside linkage between twogroups selected from Nu and LNA, or L together with G* designates a3′-terminal group; andeach LNA-L independently designates a nucleoside analogue of the generalformula I as defined above, or preferably of the general formula Ia asdefined above.

Within this embodiment, as well as generally, the present inventionprovides the intriguing possibility of including LNAs with differentnucleobases, in particular both nucleobases selected from thymine,cytosine and urasil and nucleobases selected from adenine and guanine.

In another embodiment of the present invention, the olgomer furthercomprises a PNA mono- or oligomer segment of the formula

wherein B is a defined above for the formula I, AASC designates hydrogenor an amino acid side chain, t is 1-5, and w is 1-50.

In the present context, the term “amino acid side chain” means a groupbound to the α-atom of an α-amino acids, i.e. corresponding to theα-amino acid in question without the glycine moiety, preferably aneither naturally occurring or a readily available α-amino acid.Illustrative examples are hydrogen (glycine itself), deuterium(deuterated glycine), methyl (alanine), cyanomethyl (β-cyano-alanine),ethyl, 1-propyl (norvaline), 2-propyl (valine), 2-methyl-1-propyl(leucine), 2-hydroxy-2-methyl-1-propyl (β-hydroxy-leucine), 1-butyl(norleucine), 2-butyl (isoleucine), methylthioethyl (methionine), benzyl(phenylalanine), p-amino-benzyl (p-amino-phenylalanine), p-iodo-benzyl(p-iodo-phenylalanine), p-fluoro-benzyl (p-fluoro-phenylalanine),p-bromo-benzyl (p-bromo-phenylalanine), p-chloro-benzyl(p-chloro-phenylalanine), p-nitro-benzyl (p-nitro-phenylalanine),3-pyridylmethyl (β-(3-pyridyl)-alanine), 3,5-diiodo-4-hydroxy-benzyl(3,5-diiodo-tyrosine), 3,5-dibromo-4-hydroxy-benzyl(3,5-dibromo-tyrosine), 3,5-dichloro-4-hydroxy-benzyl(3,5-dichloro-tyrosine), 3,5-difluoro-4-hydroxy-benzyl(3,5-difluoro-tyrosine), 4-methoxy-benzyl (O-methyl-tyrosine),2-naphtylmethyl (β-(2-naphtyl)-alanine), 1-naphtylmethyl(β-(1-naphtyl)-alanine), 3-indolylmethyl (tryptophan), hydroxymethyl(serine), 1-hydroxyethyl (threonine), mercaptomethyl (cysteine),2-mercapto-2-propyl (penicillamine), 4-hydroxybenzyl (tyrosine),aminocarbonylmethyl (asparagine), 2-aminocarbonylethyl (glutamine),carboxymethyl (aspartic acid), 2-carboxyethyl (glutamic acid),aminomethyl (α,β-diaminopropionic acid), 2-aminoethyl(α,γ-diaminobutyric acid), 3-amino-propyl (ornithine), 4-amino-1-butyl(lysine), 3-guanidino-1-propyl (arginine), and 4-imidazolylmethyl(histidine).

PNA mono- or oligomer segment may be incorporated in a oligomer asdescribed in EP 0672677 A2.

The oligomers of the present invention are also intended to coverchimeric oligomers. “Chimeric oligomers” means two or more oligomerswith monomers of different origin joined either directly or via aspacer. Illustrative examples of such oligomers which can be combinedare peptides, PNA-oligomers, oligomers containing LNA's, andoligonucleotide oligomers.

Apart from the oligomers defined above, the present invention alsoprovides monomeric LNAs useful, e.g., in the preparation of oligomers,as substrates for, e.g., nucleic acid polymerases, polynucleotidekinases, terminal transferases, and as therapeutical agents, see furtherbelow. The monomeric LNAs correspond in the overall structure(especially with respect to the possible biradicals) to the LNAs definedas constituents in oligomers, however with respect to the groups P andP*, the monomeric LNAs differ slightly as will be explained below.Furthermore, the monomeric LNAs may comprise functional group protectinggroups, especially in the cases where the monomeric LNAs are to beincorporated into oligomers by chemical synthesis.

An interesting subgroup of the possible monomeric LNAs comprisesbicyclic nucleoside analogues (LNAs) of the general formula II

wherein the substituent B is selected from nucleobases, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands; X is selectedfrom —O—, S—, —N(R^(N)*)—, and C(R⁶R⁶*)—, preferably from —O—, —S—, andN(R^(N)*)—; one of the substituents R², R²*, R³, and R³* is a group Q*;each of Q and Q* is independently selected from hydrogen, azido,halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—,Act-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₆-alkoxy, optionallysubstituted C₁₆-alkyl, optionally substituted C₂₆-alkenyl, optionallysubstituted C₂₆-alkenyloxy, optionally substituted C₂₆-alkynyl,optionally substituted C₂₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl;R²* and R⁴* together designate a biradical selected from —O, S, N(R*)—,(CR*R*)_(r+s+1)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, (CR*R*)_(r)—N(R*)—(CR*R*)_(s),—O—(CR*R*)_(r+s)—O—, S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)(CR*R*)_(r+s)—N(R*)—, N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; R² and R³ together designate a biradicalselected from O, (CR*R*)_(r+s)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); R²* andR³ together designate a biradical selected from O, —(CR*R*)_(r+s)—,(CR*R*)_(r)—O—(CR*R*)_(s), (CR*R*)_(r)—S—(CR*R*)_(s)—, and(CR*R*)_(r)—N(R*)—(CR*R*)_(s); R³ and R⁴* together designate a biradicalselected from (CR*R*)_(r)—O—(CR*R*)_(s)—, (CR*R*)_(r)—S—(CR*R*)_(s)—,and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); R³ and R⁵ together designate abiradical selected from (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); R¹* andR⁴* together designate a biradical selected from(CR*R*)_(r)—O—(CR*R*)_(s)—, (CR*R*)_(r)—S—(CR*R*)_(s)—, and(CR*R*)_(r)—N(R*)—(CR*R*)_(s); or R¹* and R²* together designate abiradical selected from (CR*R*)_(r)—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, and (CR*R*)_(r)—N(R*)—(CR*R*)_(s); whereinR* is as defined above for the oligomers; and each of the substituentsR¹*, R², R²*, R³, R⁴*, R⁵, and R⁵*, which are not involved in Q, Q* orthe biradical, are as defined above for the oligomers.

It should furthermore be understood, with due consideration of the knownbicyclic nucleoside analogues, that R² and R³ do not together designatea biradical selected from O—CH₂CH₂ and O—CH₂CH₂CH₂; and R³ and R⁵ do nottogether designate a biradical selected from CH₂CH₂, O—CH₂, and—O—Si(^(i)Pr)₂—O—Si(^(i)Pr)₂—O—.

The monomeric LNAs also comprise basic salts and acid addition saltsthereof. Furthermore, it should be understood that any chemical group(including any nucleobase), which is reactive under the conditionsprevailing in chemical oligonucleotide synthesis, is optionallyfunctional group protected as known in the art. This means that groupssuch as hydroxy, amino, carboxy, sulphono, and mercapto groups, as wellas nucleobases, of a monomeric LNA are optionally functional groupprotected. Protection (and deprotection) is performed by methods knownto the person skilled in the art (see, e.g., Greene, T. W. and Wuts, P.G. M., “Protective Groups in Organic Synthesis”, 2^(nd) ed., John Wiley,N.Y. (1991), and M. J. Gait, Oligonucleotide Synthesis, IRL Press,1984).

Illustrative examples of hydroxy protection groups are optionallysubstituted trityl, such as 4,4′-dimethoxytrityl (DMT),4-monomethoxytrityl (MMT), and trityl, optionally substituted9-(9-phenyl)xanthenyl (pixyl), optionally substituted ethoxycarbonyloxy,p-phenylazophenyloxycarbonyloxy, tetraahydropyranyl (thp),9-fluorenylmethoxycarbonyl (Fmoc), methoxytetrahydropyranyl (mthp),silyloxy such as trimethylsilyl (TMS), triisopropylsilyl (TIPS),tert-butyldimethylsilyl (TBDMS), triethylsilyl, and phenyldimethylsilyl,benzyloxycarbonyl or substituted benzyloxycarbonyl ethers such as2-bromo benzyloxycarbonyl, tert-butylethers, alkyl ethers such as methylether, acetals (including two hydroxy groups), acyloxy such as acetyl orhalogen substituted acetyls, e.g. chloroacetyl or fluoroacetyl,isobutyryl, pivaloyl, benzoyl and substituted benzoyls, methoxymethyl(MOM), benzyl ethers or substituted benzyl ethers such as2,6-dichlorobenzyl (2,6-Cl₂Bzl). Alternatively, the hydroxy group may beprotected by attachment to a solid support optionally through a linker.

Illustrative examples of amino protection groups are Fmoc(fluorenylmethoxycarbonyl), BOC (tert-butyloxycarbonyl),trifluoroacetyl, allyloxycarbonyl (alloc, AOC), benzyloxycarbonyl (Z,Cbz), substituted benzyloxycarbonyls such as 2-chloro benzyloxycarbonyl((2-ClZ), monomethoxytrityl (MMT), dimethoxytrityl (DMT), phthaloyl, and9-(9-phenyl)xanthenyl (pixyl).

Illustrative examples of carboxy protection groups are allyl esters,methyl esters, ethyl esters, 2-cyanoethylesters,trimethylsilylethylesters, benzyl esters (Obz1), 2-adamantyl esters(O-2-Ada), cyclohexyl esters (OcHex), 1,3-oxazolines, oxazoler,1,3-oxazolidines, amides or hydrazides.

Illustrative examples of mercapto protecting groups are trityl (Trt),acetamidomethyl (acm), trimethylacetamidomethyl (Tacm),2,4,6-trimethoxybenzyl (Tmob), tert-butylsulfenyl (StBu),9-fluorenylmethyl (Fm), 3-nitro-2-pyridinesulfenyl (Npys), and4-methylbenzyl (Meb).

Furthermore, it may be necessary or desirable to protect any nucleobaseincluded in an monomeric LNA, especially when the monomeric LNA is to beincorporated in an oligomer according to the invention. In the presentcontext, the term “protected nucleobases” means that the nucleobase inquestion is carrying a protection group selected among the groups whichare well-known for a man skilled in the art (see e.g. Protocols forOligonucleotides and Analogs, vol 20, (Sudhir Agrawal, ed.), HumanaPress, 1993, Totowa, N.J.; S. L. Beaucage and R. P. Iyer, Tetrahedron,1993, 49, 6123; S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48,2223; and E. Uhlmann and A. Peyman, Chem. Rev., 90, 543.). Illustrativeexamples are benzoyl, isobutyryl, tert-butyl, tert-butyloxycarbonyl,4-chloro-benzyloxycarbonyl, 9-fluorenylmethyl,9-fluorenylmethyloxycarbonyl, 4-methoxybenzoyl,4-methoxytriphenylmethyl, optionally substituted triazolo,p-toluenesulphonyl, optionally substituted sulphonyl, isopropyl,optionally substituted amidines, optionally substituted trityl,phenoxyacetyl, optionally substituted acyl, pixyl, tetrahydropyranyl,optionally substituted silyl ethers, and 4-methoxybenzyloxycarbonyl.Chapter 1 in “Protocols for oligonucleotide conjugates”, Methods inMolecular Biology, vol 26, (Sudhir Agrawal, ed.), Humana Press, 1993,Totowa, N.J. and S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48,2223 disclose further suitable examples.

In a preferred embodiment, the group B in a monomeric LNA is preferablyselected from nucleobases and protected nucleobases.

In an embodiment of the monomeric LNAs according to the presentinvention, one of Q and Q*, preferably Q*, designates a group selectedfrom Act-O—, Act-S—, Act-N(R^(H))—, Act-O—CH₂—, Act-S—CH₂—,Act-N(R^(H))—CH₂—, and the other of Q and Q*, preferably Q, designates agroup selected from hydrogen, azido, halogen, cyano, nitro, hydroxy,Prot-O—, mercapto, Prot-S—, C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₆-alkoxy, optionallysubstituted C₁₆-alkyl, optionally substituted C₂₆-alkenyl, optionallysubstituted C₂₆-alkenyloxy, optionally substituted C₂₆-alkynyl,optionally substituted C₂₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, aminomethyl,Prot-N(R^(H))—CH₂—, carboxymethyl, sulphonomethyl, and R^(H) is selectedfrom hydrogen and C₁₋₆-alkyl.

In the case described above, the group Prot designates a protectinggroup for —OH, —SH, and —NH(R^(H)), respectively. Such protection groupsare selected from the same as defined above for hydroxy protectiongroups, mercapto protection group, and amino protection groups,respectively, however taking into consideration the need for a stableand reversible protection group. However, it is preferred that anyprotection group for OH is selected from optionally substituted trityl,such as dimethoxytrityl (DMT), monomethoxytrityl (MMT), and trityl, and9-(9-phenyl)xanthenyl (pixyl), optionally substituted, tetrahydropyranyl(thp) (further suitable hydroxy protection groups for phosphoramiditeoligonucleotide synthesis are described in Agrawal, ed. “Protocols forOligonucleotide Conjugates”; Methods in Molecular Biology, vol. 26,Humana Press, Totowa, N.J. (1994) and Protocols for Oligonucleotides andAnalogs, vol 20, (Sudhir Agrawal, ed.), Humana Press, 1993, Totowa,N.J.), or protected as acetal; that any protection group for —SH isselected from trityl, such as dimethoxytrityl (DMT), monomethoxytrityl(MMT), and trityl, and 9-(9-phenyl)xanthenyl (pixyl), optionallysubstituted, tetrahydropyranyl (thp) (further suitable mercaptoprotection groups for phosphoramidite oligonucleotide synthesis are alsodescribed in Agrawal (see above); and that any protecting group for—NH(R^(H)) is selected from trityl, such as dimethoxytrityl (DMT),monomethoxytrityl (MMT), and trityl, and 9-(9-phenyl)xanthenyl (pixyl),optionally substituted, tetrahydropyranyl (thp) (further suitable aminoprotection groups for phosphoramidite oligonucleotide synthesis are alsodescribed in Agrawal (see above).

In the embodiment above, as well as for any monomeric LNAs definedherein, Act designates an activation group for —OH, —SH, and —NH(R^(H)),respectively. Such activation groups are, e.g., selected from optionallysubstituted O-phosphoramidite, optionally substitutedO-phosphortriester, optionally substituted O-phosphordiester, optionallysubstituted H-phosphonate, and optionally substituted O-phosphonate.

In the present context, the term “phosphoramidite” means a group of theformula P(OR^(x))—N(R^(y))₂, wherein R^(x) designates an optionallysubstituted alkyl group, e.g. methyl, 2cyanoethyl, or benzyl, and eachof R^(y) designate optionally substituted alkyl groups, e.g. ethyl orisopropyl, or the group —N(R^(y))₂ forms a morpholino group(N(CH₂CH₂)₂O). R^(x) preferably designates 2-cyanoethyl and the twoR^(y) are preferably identical and designate isopropyl. Thus, anespecially relevant phosphoramidite is N,Ndiisopropyl-O(2cyanoethyl)phosphoramidite.

It should be understood that the protecting groups used herein for asingle monomeric LNA or several monomeric LNAs may be selected so thatwhen this/these LNA(s) are incorporated in an oligomer according to theinvention, it will be possible to perform either a simultaneousdeprotection or a sequential deprotection of the functional groups. Thelatter situation opens for the possibility of regioselectivelyintroducing one or several “active/functional” groups such as DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, where suchgroups may be attached via a spacer as described above.

In a preferred embodiment, Q is selected from hydrogen, azido, halogen,cyano, nitro, hydroxy, Prot-O—, mercapto, Prot-S—, C₁₆-alkylthio, amino,Prot-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionally substitutedC₁₆-alkoxy, optionally substituted C₁₆-alkyl, optionally substitutedC₂₆-alkenyl, optionally substituted C₂₆-alkenyloxy, optionallysubstituted C₂₆-alkynyl, optionally substituted C₂₆-alkynyloxy,monophosphate, diphosphate, triphosphate, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl,Prot-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl; and Q* is selected from hydrogen, azido, halogen, cyano,nitro, hydroxy, Act-O—, mercapto, ActS, C₁₋₆-alkylthio, amino,Act-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino, optionally substitutedC₁₆-alkoxy, optionally substituted C₁₆-alkyl, optionally substitutedC₂₆-alkenyl, optionally substituted C₂₆-alkenyloxy, optionallysubstituted C₂₆-alkynyl, optionally substituted C₂₆-alkynyloxy, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, ligands, carboxy, sulphono,where Act is an activation group for —OH, —SH, and —NH(R^(H)),respectively, and R^(H) is selected from hydrogen and C₁₋₆-alkyl.

The monomeric LNAs of the general formula II may, as the LNAsincorporated into oligomers, represent various stereoisomers. Thus, thestereochemical variants described above for the LNAs incorporated intooligomers are believed to be equally applicable in the case of monomericLNAs (however, it should be noted that P should then be replaced withQ).

In a preferred embodiment of the present invention, the monomeric LNAhas the general formula IIa

wherein the substituents are defined as above.

Furthermore, with respect to the definitions of substituents,biradicals, R*, etc. the same preferred embodiments as defined above forthe oligomer according to the invention also apply in the case ofmonomeric LNAs.

In a particularly interesting embodiment of the monomeric LNAs of thepresent invention, B designates a nucleobase, preferably a nucleobaseselected from thymine, cytosine, urasil, adenine and guanine (inparticular adenine and guanine), X is —O—, R²* and R⁴* togetherdesignate a biradical selected from (CH₂)₀₋₁—O(CH₂)₁₋₃—,(CH₂)₀₋₁—S(CH₂)₁₃—, and (CH₂)₀₋₁—N(R^(N))(CH₂)₁₋₃—, in particular—O—CH₂—, —S—CH₂— and —R^(N)—CH₂—, where R^(N) is selected from hydrogenand C₁₋₄-alkyl, Q designates Prot-O—, R³* is Q* which designates Act-OH,and R¹*, R², R³, R⁵, and R⁵* each designate hydrogen. In thisembodiment, R^(N) may also be selected from DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups and ligands.

In a further particularly interesting embodiment of the monomeric LNAsof the present invention, B designates a nucleobase, preferably anucleobase selected from thymine, cytosine, urasil, adenine and guanine(in particular adenine and guanine), X is —O—, R²* and R⁴* togetherdesignate a biradical selected from (CH₂)₀₋₁—O(CH₂)₁₋₃—,(CH₂)₀₋₁—S(CH₂)₁₋₃—, and (CH₂)₀₋₁—N(R^(N))(CH₂)₁₋₃—, in particular—O—CH₂—, —S—CH₂— and —R^(N)—CH₂—, where R^(N) is selected from hydrogenand C₁₋₄-alkyl, Q is selected from hydroxy, mercapto, C₁₋₆-alkylthio,amino, mono- or di(C₁₋₆-alkyl)amino, optionally substituted C₁₆-alkoxy,optionally substituted C₂₆-alkenyloxy, optionally substitutedC₂₆-alkynyloxy, monophosphate, diphosphate, and triphosphate, R³* is Q*which is selected from hydrogen, azido, halogen, cyano, nitro, hydroxy,mercapto, C₁₋₆-alkylthio, amino, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₆-alkoxy, optionally substituted C₁₆-alkyl,optionally substituted C₂₆-alkenyl, optionally substitutedC₂₆-alkenyloxy, optionally substituted C₂₆-alkynyl, and optionallysubstituted C₂₆-alkynyloxy, R³ is selected from hydrogen, optionallysubstituted C₁₆-alkyl, optionally substituted C₂₆-alkenyl, andoptionally substituted C₂₆-alkynyl, and R¹*, R², R⁵, and R⁵* eachdesignate hydrogen. Also here, R^(N) may also be selected from DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups and ligands.

In a further particularly interesting embodiment of the monomeric LNAsof the present invention, B designates a nucleobase, X is —O—, R² and R³together designate a biradical selected from (CH₂)₀₋₁—OCH═CH—,(CH₂)₀₋₁—SCH═CH—, and (CH₂)₀₋₁—N(R^(N))CH═CH— where R^(N) is selectedfrom hydrogen and C₁₋₄-alkyl, Q is selected from hydroxy, mercapto,C₁₋₆-alkylthio, amino, mono- or di(C₁₋₆-alkyl)amino, optionallysubstituted C₁₆-alkoxy, optionally substituted C₂₆-alkenyloxy,optionally substituted C₂₆-alkynyloxy, monophosphate, diphosphate, andtriphosphate, R³* is Q* which is selected from hydrogen, azido, halogen,cyano, nitro, hydroxy, mercapto, C₁₋₆-alkylthio, amino, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₆-alkoxy, optionallysubstituted C₁₆-alkyl, optionally substituted C₂₆-alkenyl, optionallysubstituted C₂₆-alkenyloxy, optionally substituted C₂₆-alkynyl, andoptionally substituted C₂₆-alkynyloxy, and R¹*, R²*, R⁴*, R⁵, and R⁵*each designate hydrogen.

One aspect of the invention is to provide various derivatives of LNAsfor solid-phase and/or solution phase incorporation into an oligomer. Asan illustrative example, monomers suitable for incorporation of(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxy-methyl-3-(cytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(urasil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(guanin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,and(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(adenin-1-yl)-2,5-dioxabicyclo[2.2.1]heptaneusing the phosphoramidite approach, the phosphortriester approach, andthe H-phosphonate approach, respectively, are(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1R,3R,4R,7S)-7-hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane-7-O-(2-chlorophenylphosphate),and(1R,3R,4R,7S)-7-hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane-7-O—(H-phosphonate)and the 3-(cytosin-1-yl), 3-(urasil-1-yl), 3-(adenin-1-yl) and3-(guanin-1-yl) analogues thereof, respectively. Furthermore, theanalogues where the methyleneoxy biradical of the monomers issubstituted with a methylenethio, a methyleneamino, or a 1,2-ethylenebiradical are also expected to constitute particularly interestingvariants within the present invention. The methylenethio andmethyleneamino analogues are believed to equally applicable as themethyleneoxy analogue and therefore the specific reagents correspondingto those mentioned for incorporation of(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(cytosin-1-yl)-2,5-dioxabicyclo[2.2.1A]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(urasil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(guanin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,and(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(adenin-1-yl)-2,5-dioxabicyclo[2.2.1]heptaneshould also be considered as particularly interesting reactive monomerswithin the present invention. For the methyleneamine analogue, it shouldbe noted that the secondary amine may carry a substituent selected fromoptionally substituted C₁₋₆-alkyl such as methyl and benzyl, optionallysubstituted C₁₋₆-alkylcarbonyl such as trifluoroacetyl, optionallysubstituted arylcarbonyl and optionally substituted heteroarylcarbonyl.

In a particularly interesting embodiment, the present invention relatesto an oligomer comprising at least one LNA of the general formula Ia

wherein X is —O—; B is selected from nucleobases, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands; P designates the radical positionfor an internucleoside linkage to a succeeding monomer, or a 5′-terminalgroup, such internucleoside linkage or 5′-terminal group optionallyincluding the substituent R⁵; R³* is a group P* which designates aninternucleoside linkage to a preceding monomer, or a 3′-terminal group;R²* and R⁴* together designate a biradical selected from −O, S, N(R*),(CR*R*)_(r+s+1)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, (CR*R*)_(r)—N(R*)—(CR*R*)_(s),—O—(CR*R*)_(r+s)—O—, S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)(CR*R)_(r+s)—N(R*)—, —N(R)—(CR*R)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; wherein each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond, and each of r and s is 0-3 with the provisothat the sum r+s is 1-4; each of the substituents R¹*, R², R³, R⁵, andR⁵* is independently selected from hydrogen, optionally substitutedC₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy, C₁₋₆-alkoxy,C₂₋₆-alkenyloxy, carboxy, C₁₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl,formyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, C₁₋₆-alkyl-carbonylamino, carbamido,azido, C₁₋₆-alkanoyloxy, sulphono, sulphanyl, C₁₋₆-alkylthio, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, and halogen,where two geminal substituents together may designate oxo; and basicsalts and acid addition salts thereof. In particular, one R* is selectedfrom hydrogen, hydroxy, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and any remaining substituents R* are hydrogen.Especially, the biradical is selected from —O—, (CH₂)₀₋₁—O(CH₂)₁₋₃—,(CH₂)₀₋₁—S(CH₂)₁₋₃—, (CH₂)₀₋₁—N(R^(N))(CH₂)₁₋₃—, and (CH₂)₂₋₄—.

In a further particularly interesting embodiment, the present inventionrelates to an LNA of the general formula IIa

wherein X is —O—; B is selected from nucleobases, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands; R³* is a group Q*; each of Q andQ* is independently selected from hydrogen, azido, halogen, cyano,nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—, Act-S—,C₁₋₆-alkylthio, amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- ordi(C₁₋₆-alkyl)amino, optionally substituted C₁₆-alkoxy, optionallysubstituted C₁₆-alkyl, optionally substituted C₂₆-alkenyl, optionallysubstituted C₂₆-alkenyloxy, optionally substituted C₂₆-alkynyl,optionally substituted C₂₆-alkynyloxy, monophosphate, diphosphate,triphosphate, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups,ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH₂—, Act-O—CH₂—,aminomethyl, Prot-N(R^(H))—CH₂—, Act-N(R^(H))—CH₂—, carboxymethyl,sulphonomethyl, where Prot is a protection group for —OH, —SH, and—NH(R^(H)), respectively, Act is an activation group for —OH, —SH, and—NH(R^(H)), respectively, and R^(H) is selected from hydrogen andC₁₋₆-alkyl; R²* and R⁴* together designate a biradical selected from −O,S, N(R*), (CR*R*)_(r+s+1)—, (CR*R*)_(r)—O—(CR*R*)_(s)—,(CR*R*)_(r)—S—(CR*R*)_(s)—, (CR*R*)_(r)—N(R*)—(CR*R*)_(s),—O—(CR*R*)_(r+s)—O—, S—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—S—,N(R*)—(CR*R*)_(r+s)—O—, —O—(CR*R*)_(r+s)—N(R*)—, —S—(CR*R*)_(r+s)—S—,—N(R*)(CR*R*)_(r+s)—N(R*)—, —N(R*)—(CR*R*)_(r+s)—S—, and—S—(CR*R*)_(r+s)—N(R*)—; wherein each R* is independently selected fromhydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono-or di(C₁₋₆-alkyl)amino, optionally substituted C₁₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and/or two adjacent (non-geminal) R* may togetherdesignate a double bond, and each of r and s is 0-3 with the provisothat the sum r+s is 1-4; each of the substituents R¹*, R², R³, R⁵, andR⁵* is independently selected from hydrogen, optionally substitutedC₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy, C₁₋₆-alkoxy,C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl,formyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, C₁₋₆-alkyl-carbonylamino, carbamido,azido, C₁₋₆-alkanoyloxy, sulphono, sulphanyl, C₁₋₆-alkylthio, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, and halogen,where two geminal substituents together may designate oxo; and basicsalts and acid addition salts thereof; and with the proviso that anychemical group (including any nucleobase), which is reactive under theconditions prevailing in oligonucleotide synthesis, is optionallyfunctional group protected. Preferably, one R* is selected fromhydrogen, hydroxy, optionally substituted C₁₋₆-alkoxy, optionallysubstituted C₁₋₆-alkyl, DNA intercalators, photochemically activegroups, thermochemically active groups, chelating groups, reportergroups, and ligands, and any remaining substituents R* are hydrogen.Especially, the biradical is selected from —O—, (CH₂)₀₋₁—O(CH₂)₁₋₃—,(CH₂)₀₋₁—S(CH₂)₁₋₃—, (CH₂)₀₋₁—N(R^(N))(CH₂)₁₋₃—, and (CH₂)₂₋₄—.

Generally, the present invention provides oligomers having surprisinglygood hybridisation properties with respect to affinity and specificity.Thus, the present invention provides an oligomer comprising at least onenucleoside analogue which imparts to the oligomer a T_(m) with acomplementary DNA oligonucleotide which is at least 2.5° C. higher,preferably at least 3.5° C. higher, in particular at least 4.0° C.higher, especially at least 5.0° C. higher, than that of thecorresponding unmodified reference oligonucleotide which does notcomprise any nucleoside analogue. In particular, the T_(m) of theoligomer is at least 2.5×N ° C. higher, preferably at least 3.5×N ° C.higher, in particular at least 4.0×N ° C. higher, especially at least5.0×N° C. higher, where N is the number of nucleoside analogues.

In the case of hybridisation with a complementary RNA oligonucleotide,the at least one nucleoside analogue imparts to the oligomer a T_(m)with the complementary DNA oligonucleotide which is at least 4.0° C.higher, preferably at least 5.0° C. higher, in particular at least 6.0°C. higher, especially at least 7.0° C. higher, than that of thecorresponding unmodified reference oligonucleotide which does notcomprise any nucleoside analogue. In particular, the T_(m) of theoligomer is at least 4.0×N ° C. higher, preferably at least 5.0×N ° C.higher, in particular at least 6.0×N ° C. higher, especially at least7.0×N° C. higher, where N is the number of nucleoside analogues.

The term “corresponding unmodified reference oligonucleotide” isintended to mean an oligonucleotide solely consisting of naturallyoccurring nucleotides which represents the same nucleobases in the sameabsolute order (and the same orientation).

The T_(m) is measured under one of the following conditions (i.e.essentially as illustrated in Example 129):

a) 10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl, 0.1 mM EDTA;

b) 10 mM Na₂HPO₄ pH 7.0, 0.1 mM EDTA; or

c) 3M tetramethylammoniumchlorid (TMAC), 10 mM Na₂HPO₄, pH 7.0, 0.1 mMEDTA;

preferably under conditions a), at equimolar amounts (typically 1.0 μM)of the oligomer and the complementary DNA oligonucleotide.

The oligomer is preferably as defined above, where the at least onenucleoside analogue has the formula I where B is a nucleobase. Inparticular interesting is the cases where at least one nucleosideanalogue includes a nucleobase selected from adenine and guanine.

Furthermore, with respect to specificity and affinity, the oligomer,when hybridised with a partially complementary DNA oligonucleotide, or apartially complementary RNA oligonucleotide, having one or moremismatches with said oligomer, should exhibit a reduction in T_(m), as aresult of said mismatches, which is equal to or greater than thereduction which would be observed with the corresponding unmodifiedreference oligonucleotide which does not comprise any nucleosideanalogues. Also, the oligomer should have substantially the samesensitivity of T_(m) to the ionic strength of the hybridisation bufferas that of the corresponding unmodified reference oligonucleotide.

Oligomers defined herein are typically at least 30% modified, preferablyat least 50% modified, in particular 70% modified, and in someinteresting applications 100% modified.

The oligomers of the invention has substantially higher3′-exonucleolytic stability than the corresponding unmodified referenceoligonucleotide. This important property can be examined as described inExample 136.

DEFINITIONS

In the present context, the term “C₁₋₁₂-alkyl” means a linear, cyclic orbranched hydrocarbon group having 1 to 12 carbon atoms, such as methyl,ethyl, propyl, iso-propyl, cyclopropyl, butyl, tert-butyl, iso-butyl,cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and dodecyl.Analogously, the term “C₁₆-alkyl” means a linear, cyclic or branchedhydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl,propyl, iso-propyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the term“C₁₋₄-alkyl” is intended to cover linear, cyclic or branched hydrocarbongroups having 1 to 4 carbon atoms, e.g. methyl, ethyl, propyl,iso-propyl, cyclopropyl, butyl, iso-butyl, tert-butyl, cyclobutyl.

Preferred examples of “C₁₋₆-alkyl” are methyl, ethyl, propyl,iso-propyl, butyl, tert-butyl, iso-butyl, pentyl, cyclopentyl, hexyl,cyclohexyl, in particular methyl, ethyl, propyl, iso-propyl, tert-butyl,iso-butyl and cyclohexyl. Preferred examples of “C₁₋₄-alkyl” are methyl,ethyl, propyl, iso-propyl, butyl, tert-butyl, and iso-butyl.

Similarly, the term “C₂₋₁₂-alkenyl” covers linear, cyclic or branchedhydrocarbon groups having 2 to 12 carbon atoms and comprising oneunsaturated bond. Examples of alkenyl groups are vinyl, allyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, dodecaenyl. Analogously, the term“C₂₋₆-alkenyl” is intended to cover linear, cyclic or branchedhydrocarbon groups having 2 to 6 carbon atoms and comprising oneunsaturated bond. Preferred examples of alkenyl are vinyl, allyl,butenyl, especially allyl.

Similarly, the term “C₂₋₁₂-alkynyl” means a linear or branchedhydrocarbon group having 2 to 12 carbon atoms and comprising a triplebond. Examples hereof are ethynyl, propynyl, butynyl, octynyl, anddodecanyl.

In the present context, i.e. in connection with the terms “alkyl”,“alkenyl”, and “alkynyl”, the term “optionally substituted” means thatthe group in question may be substituted one or several times,preferably 1-3 times, with group(s) selected from hydroxy (which whenbound to an unsaturated carbon atom may be present in the tautomericketo form), C₁₋₆-alkoxy (i.e. C₁₋₆-alkyl-oxy), C₂₋₆-alkenyloxy, carboxy,oxo (forming a keto or aldehyde functionality), C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono and di(C₁₋₆-alkyl)amino; carbamoyl,mono- and di(C₁₋₆-alkyl)aminocarbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,mono and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkylcarbonylamino, cyano, guanidino, carbamido, C₁₋₆-alkanoyloxy,sulphono, C₁₋₆-alkylsulphonyloxy, nitro, sulphanyl, C₁₋₆-alkylthio,halogen, where any aryl and heteroaryl may be substituted asspecifically describe below for “optionally substituted aryl andheteroaryl”.

Preferably, the substituents are selected from hydroxy, C₁₋₆-alkoxy,carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl, aryl,aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono anddi(C₁₋₆-alkyl)amino, carbamoyl, mono- and di(C₁₋₆-alkyl)aminocarbonyl,amino-C₁₋₆-alkyl-aminocarbonyl, mono anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkylcarbonylamino,cyano, carbamido, halogen, where aryl and heteroaryl may be substituted1-5 times, preferably 1-3 times, with C₁₋₄-alkyl, C₁₋₄-alkoxy, nitro,cyano, amino or halogen. Especially preferred examples are hydroxy,C₁₋₆-alkoxy, carboxy, aryl, heteroaryl, amino, mono anddi(C₁₋₆-alkyl)amino, and halogen, where aryl and heteroaryl may besubstituted 1-3 times with C₁₋₄-alkyl, C₁₋₄-alkoxy, nitro, cyano, aminoor halogen.

In the present context the term “aryl” means a fully or partiallyaromatic carbocyclic ring or ring system, such as phenyl, naphthyl,1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl,benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferredexample.

The term “heteroaryl” means a fully or partially aromatic carbocyclicring or ring system where one or more of the carbon atoms have beenreplaced with heteroatoms, e.g. nitrogen (═N— or NH), sulphur, and/oroxygen atoms. Examples of such heteroaryl groups are oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl,pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl,quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzooxozolyl,phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl,carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl, phenoxazonyl.

In the present context, i.e. in connection with the terms “aryl” and“heteroaryl”, the term “optionally substituted” means that the group inquestion may be substituted one or several times, preferably 1-5 times,in particular 1-3 times) with group(s) selected from hydroxy (which whenpresent in an enol system may be represented in the tautomeric ketoform), C₁₋₆-alkyl, C₁₋₆-alkoxy, oxo (which may be represented in thetautomeric enol form), carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl,formyl, aryl, aryloxy, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino,mono and di(C₁₋₆-alkyl)amino; carbamoyl, mono- anddi(C₁₋₆-alkyl)aminocarbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkylcarbonylamino,cyano, guanidino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, sulphanyl, dihalogen-C₁₋₄-alkyl,trihalogen-C₁₋₄-alkyl, halogen, where aryl and heteroaryl representingsubstituents may be substituted 1-3 times with C₁₋₄-alkyl, C₁₋₄-alkoxy,nitro, cyano, amino or halogen. Preferred examples are hydroxy,C₁₋₆-alkyl, C₁₋₆-alkoxy, carboxy, C₁₋₆-alkoxycarbonyl,C₁₋₆-alkylcarbonyl, aryl, amino, mono and di(C₁₋₆-alkyl)amino, andhalogen, wherein aryl may be substituted 1-3 times with C₁₋₄-alkyl,C₁₋₄-alkoxy, nitro, cyano, amino or halogen.

“Halogen” includes fluoro, chloro, bromo, and iodo.

It should be understood that oligomers (wherein LNAs are incorporated)and LNAs as such include possible salts thereof, of whichpharmaceutically acceptable salts are especially relevant. Salts includeacid addition salts and basic salts. Examples of acid addition salts arehydrochloride salts, sodium salts, calcium salts, potassium salts, etc.Examples of basic salts are salts where the (remaining) counter ion isselected from alkali metals, such as sodium and potassium, alkalineearth metals, such as calcium, and ammonium ions (⁺N(R^(g))₃R^(h), whereeach of R^(g) and R^(h) independently designates optionally substitutedC₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionally substitutedaryl, or optionally substituted heteroaryl). Pharmaceutically acceptablesalts are, e.g., those described in Remington's Pharmaceutical Sciences,17. Ed. Alfonso R. Gennaro (Ed.), Mack Publishing Company, Easton, Pa.,U.S.A., 1985 and more recent editions and in Encyclopedia ofPharmaceutical Technology. Thus, the term “an acid addition salt or abasic salt thereof” used herein is intended to comprise such salts.Furthermore, the oligomers and LNAs as well as any intermediates orstarting materials therefor may also be present in hydrate form.

Preparation of Monomers

In a preferred embodiment, nucleosides containing an additional2′-0,4′-C-linked ring were synthesised by the following procedure:

Synthesis of a number of 4′-C-hydroxymethyl nucleosides have beenreported earlier (R. D. Youssefyeh, J. P. H. Verheyden and J. G.Moffatt, J. Org. Chem., 1979, 44, 1301; G. H. Jones, M. Taniguchi, D.Tegg and J. G. Moffatt, J. Org. Chem., 1979, 44, 1309; C. O-Yang, H. Y.Wu, E. B. Fraser-Smith and K. A. M. Walker, Tetrahedron Lett., 1992, 33,37; H. Thrane, J. Fensholdt, M. Regner and J. Wengel, Tetrahedron, 1995,51, 10389; K. D. Nielsen, F. Kirpekar, P. Roepstorff and J. Wengel,Bioorg. Med. Chem., 1995, 3, 1493). For exemplification of synthesis of2′-O,4′-C-linked bicyclic nucleosides we chose a strategy starting from4′-C-hydroxymethyl furanose derivative 31. Benzylation, acetylation, andacetolysis followed by another acetylation afforded furanose 33, a keyintermediate for nucleoside coupling. Stereoselective reaction withsilylated thymine afforded compound 34 which was deacetylated to givenucleoside diol 35. Tosylation followed by base-induced ring closureafforded the 2′-O,4′-C-linked bicyclic nucleoside derivative 36.Debenzylation yielded the unprotected bicyclic nucleoside analogue 37which was transformed into the 5′-O-4,4′-dimethoxytrityl protectedanalogue 38 and subsequently into the phosphoramidite derivative 39 foroligonucleotide synthesis. A similar procedure has been used forsynthesis of the corresponding uracil, adenine, cytosine and guaninenucleosides as exemplified in the example section. This coupling methodis only one of several possible as will be apparent for a person skilledin the art. A strategy starting from a preformed nucleoside is alsopossible. Thus, for example, conversion of uridine derivative 62 toderivative 44 was successfully accomplished by tosylation,deisopropylidination and base-induced ring-closure. As another example,conversion of nucleoside 67 into nucleoside 61B has been accomplished asdepicted in FIG. 34. Conversion of the bicyclic thymine nucleoside 37into the corresponding 5-methyl-cytosine nucleoside 65 was accomplishedby a known reaction sequence using triazole and POCl₃ followed bybenzoylation and treatment by ammonia. A similar procedure should beapplicable for the synthesis of 57C from 44. As another example ofpossible strategies, coupling of precyclised furanose derivativesalready containing an additional ring with nucleobase derivatives ispossible. Such a strategy would in addition allow preparation of thecorresponding α-nucleoside analogues. When coupling with a protectedmethyl furanoside of 4-C,2-O-methylene-D-ribofuranose, we obtainedmainly a ring-opened product. However, coupling of 1-O-acetyl furanose207 or thiophenyl furanose 212 yielded successfully LNA nucleosides withthe α-anomers as one product. Incorporation of such α-LNA nucleosideswill be possible using the standard oligomerisation techniques (as forthe LNA oligomers containing Z) yielding α-LNA oligomers. In addition, asynthetic strategy performing nucleoside coupling using a4′-C-hydroxymethyl furanose already activated for ring closure (e.g. bycontaining a mesyl or tosyl group at the 4′-C-hydroxymethyl group), ispossible as exemplified by conversion of furanose 78 to nucleoside 79followed by deprotection and ring closure to give 36. Chemical orenzymatic transglycosylation or anomerisation of appropriate furanosederivatives or nucleosides are yet other possible synthetic strategies.These and other related strategies allow for synthesis of bicyclicnucleosides containing other nucleobases or analogues thereof by eithercoupling with these nucleobases or analogues, or starting from preformednucleoside derivatives.

The described examples are meant to be illustrative for the proceduresand examples of this invention. The structures of the synthesisedcompounds were verified using 1D or 2D NMR techniques, e.g. NOEexperiments.

An additional embodiment of the present invention is to provide bicyclicnucleosides containing additional rings of different sizes and ofdifferent chemical structures. From the methods described it is obviousfor a person skilled in the art of organic synthesis that cyclisation ofother nucleosides is possible using similar procedures, also ofnucleosides containing different C-branches. The person skilled in theart will be able to find inspiration and guidance for the preparation ofsubstituted nucleoside analogue intermediates in the literature, seee.g. WO 96/14329. Regarding rings of different chemical compositions itis clear that using similar procedures or procedures well-established inthe field of organic chemistry, synthesis of for example thio analoguesof the exemplified oxo analogues is possible as is the synthesis of thecorresponding amino analogues (using for example nucleophilicsubstitution reactions or reductive alkylations).

In the example section, synthesis of the amino LNA analogues 73-74F aredescribed. Conversion of 74 and 74D into standard building blocks foroligomerisation was possible by 5′-O-DMT protection and3′-O-phosphitylation following the standard procedures. For the aminoLNA analogue, protection of the 2′-amino functionality is needed forcontrolled linear oligomerisation. Such protection can be accomplishedusing standard amino group protection techniques like, e.g., Fmoc,trifluoroacetyl or BOC. Alternatively, an N-alkyl group (e.g. benzyl,methyl, ethyl, propyl or functionalised alkyl) can be kept on duringnucleoside transformations and oligomerisation. In FIGS. 35 and 36,strategies using N-trifluoroacetyl and N-methyl derivatives are shown.As outlined in FIG. 37, conversion of nucleoside 75 into the 2′-thio-LNAnucleoside analogue 76D has been successfully performed as has thesubsequent syntheses of the phosphoramidite derivative 76F. Compound 76Fhas the required structure for automated synthesis of 2′-thio-LNAoligonucleotides. The N-trifluoroacetyl 2′-amino-LNA synthon 74A has therequired structure for automated synthesis of 2′-amino-LNAoligonucleotides.

Synthesis of the corresponding cytosine, guanine, and adeninederivatives of the 2′-thio and 2′-amino LNA nucleosides can beaccomplished using strategies analogous to those shown in FIGS. 35, 36and 37. Alternative, the stereochemistry around C-2′ can be invertedbefore cyclisations either by using a conveniently configurated, e.g. anarabino-configurated, furanose synthon, or by inverting theconfiguration around the C2′ carbon atom starting from aribo-configurated nucleoside/furanose. Subsequent activation of the2′-β-OH, e.g. by tosylation, double nucleophilic substitution as in theurasil/thymine example described above, could furnish the desiredbicyclic 2′-thio-LNA or 2′-amino-LNA nucleosides. The thus obtainedproperly protected cytosine, guanine, and adenine analogues can beprepared for oligomerisation using the standard reactions(DMT-protection and phosphitylation) as described above for otherexamples.

Preparation of Oligomers

Linear-, branched- (M. Grøtli and B. S. Sproat, J. Chem. Soc., Chem.Commun., 1995, 495; R. H. E. Hudson and M. J. Damha, J. Am. Chem. Soc.,1993, 115, 2119; M. Von Büren, G. V. Petersen, K. Rasmussen, G.Brandenburg, J. Wengel and F. Kirpekar, Tetrahedron, 1995, 51, 8491) andcircular- (G. Prakash and E. T. Kool, J. Am. Chem. Soc., 1992, 114,3523) Oligo- and polynucleotides of the invention may be produced usingthe polymerisation techniques of nucleic acid chemistry well known to aperson of ordinary skill in the art of organic chemistry.Phosphoramidite chemistry (S. L. Beaucage and R. P. Iyer, Tetrahedron,1993, 49, 6123; S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48,2223) was used, but e.g. H-phosphonate chemistry, phosphortriesterchemistry or enzymatic synthesis could also be used. Generally, standardcoupling conditions and the phosphoramidite approach was used, but forsome monomers of the invention longer coupling time, and/or repeatedcouplings with fresh reagents, and/or use of more concentrated couplingreagents were used. As another possibility, activators more active than1H-tetrazole could also be used to increase the rate of the couplingreaction. The phosphoramidietes 39, 46, 53, 57D, 61D, and 66 all coupledwith satisfactory >95% step-wise coupling yields. Anall-phosphorothioate LNA oligomer (Table 7) was synthesised usingstandard procedures. Thus, by exchanging the normal, e.g.iodine/pyridine/H₂O, oxidation used for synthesis of phosphordiesteroligomers with an oxidation using Beaucage's reagent (commerciallyavailable), the phosphorthioate LNA oligomer was efficiently synthesised(stepwise coupling yields >=98%). The 2′-amino-LNA and 2′methylamino-LNA oligonucleotides (Table 9) were efficiently synthesised(step-wise coupling yields 98%) using amidites 74A and 74F. The2′-thio-LNA oligonucleotides (Table 8) were efficiently synthesisedusing amidite 76F using the standard phosphoramidite procedures asdescribed above for LNA oligonucleotides. After synthesis of the desiredsequence, work up was done using standard conditions (cleavage fromsolid support and removal of protection groups using 30% ammonia for 55°C. for 5 h). Purification of LNA oligonucleotides was done usingdisposable reversed phase purification cartridges and/or reversed phaseHPLC and/or precipitation from ethanol or butanol. Capillary gelelectrophoresis, reversed phase HPLC and MALDI-MS was used to verify thepurity of the synthesised oligonucleotide analogues, and to verify thatthe desired number of bicyclic nucleoside analogues of the inventionwere incorporated as contemplated.

An additional aspect of the present invention is to furnish proceduresfor oligonucleotide analogues containing LNA linked by non-naturalinternucleoside linkages. For example, synthesis of the correspondingphosphorothioate or phosphoramidate analogues is possible usingstrategies well-established in the field of oligonucleotide chemistry(Protocols for Oligonucleotides and Analogs, vol 20, (Sudhir Agrawal,ed.), Humana Press, 1993, Totowa, N.J.; S. L. Beaucage and R. P. Iyer,Tetrahedron, 1993, 49, 6123; S. L. Beaucage and R. P. Iyer, Tetrahedron,1992, 48, 2223; E. Uhlmann and A. Peyman, Chem. Rev., 90, 543).

Thus, generally the present invention also provides the use of an LNA asdefined herein for the preparation of an LNA modified oligonucleotides.Is should be understood that LNA modified oligonucleotide may comprisenormal nucleosides (i.e. naturally occurring nucleosides such asribonucleosides and/or dioxyribonucleosides), as well as modifiednucleosides different from those defined with the general formula II. Ina particularly interesting embodiment, incorporation of LNA modulatesthe ability of the oligonucleotide to act as a substrate for nucleicacid active enzymes.

Furthermore, solid support materials having immobilised thereto anoptionally nucleobase protected and optionally 5′-OH protected LNA areespecially interesting as material for the synthesis of LNA modifiedoligonucleotides where an LNA monomer is included in at the 3′ end. Inthis instance, the solid support material is preferable CPG, e.g. areadily (commercially) available CPG material onto which a3′-functionalised, optionally nucleobase protected and optionally 5′-OHprotected LNA is linked using the conditions stated by the supplier forthat particular material. BioGenex Universial CPG Support (BioGenex,U.S.A.) can e.g. be used. The 5′-OH protecting group may, e.g., be a DMTgroup. 3′-functional group should be selected with due regard to theconditions applicable for the CPG material in question.

Applications

The present invention discloses the surprising finding that variousnovel derivatives of bicyclic nucleoside monomers (LNAs), whenincorporated into oligonucleotides, dramatically increase the affinityof these modified oligonucleotides for both complementary ssDNA andssRNA compared to the unmodified oligonucleotides. It further disclosesthe surprising finding that both fully and partly LNA modifiedoligonucleotides display greatly enhanced hybridisation properties fortheir complementary nucleic acid sequences. Depending on theapplication, the use of these LNAs thus offers the intriguingpossibility to either greatly increase the affinity of a standardoligonucleotide without compromising specificity (constant size ofoligonucleotide) or significantly increase the specificity withoutcompromising affinity (reduction in the size of the oligonucleotide).The present invention also discloses the unexpected finding that LNAmodified oligonucleotides, in addition to greatly enhanced hybridisationproperties, display many of the useful physicochemical properties ofnormal DNA and RNA oligonucleotides. Examples given herein includeexcellent solubility, a response of LNA modified oligonucleotides tosalts like sodium chloride and tetramethylammonium chloride which mimicthat of the unmodified oligonucleotides, the ability of LNA modifiedoligonucleotides to act as primers for a variety of polymerases, theability of LNA modified nucleotides to act as primers in a targetamplification reaction using a thermostable DNA polymerase, the abilityof LNA modified oligonucleotides to act as a substrate for T4polynucleotide kinase, the ability of biotinylated LNAs to sequencespecifically capture PCR amplicons onto a streptavidine coated solidsurface, the ability of immobilised LNA modified oligonucleotides tosequence specifically capture amplicons and very importantly the abilityof LNA modified oligonucleotides to sequence specifically targetdouble-stranded DNA by strand invasion. Hence, it is apparent to one ofordinary skills in the art that these novel nucleoside analogues areextremely useful tools to improve the performance in general ofoligonucleotide based techniques in therapeutics, diagnostics andmolecular biology.

An object of the present invention is to provide monomeric LNAsaccording to the invention which can be incorporated intooligonucleotides using procedures and equipment well known to oneskilled in the art of oligonucleotide synthesis.

Another object of the present invention is to provide fully or partlyLNA modified oligonucleotides (oligomers) that are able to hybridise ina sequence specific manner to complementary oligonucleotides formingeither duplexes or triplexes of substantially higher affinity than thecorresponding complexes formed by unmodified oligonucleotides.

Another object of the present invention is to use LNAs to enhance thespecificity of normal oligonucleotides without compromising affinity.This can be achieved by reducing the size (and therefore affinity) ofthe normal oligonucleotide to an extent that equals the gain in affinityresulting from the incorporation of LNAs.

Another object of the present invention is to provide fully or partlymodified oligonucleotides containing both LNAs, normal nucleosides andother nucleoside analogues.

A further object of the present invention is to exploit the highaffinity of LNAs to create modified oligonucleotides of extreme affinitythat are capable of binding to their target sequences in a dsDNAmolecule by way of “strand displacement”.

A further object of the invention is to provide different classes ofLNAs which, when incorporated into oligonucleotides, differ in theiraffinity towards their complementary nucleosides. In accordance with theinvention this can be achieved by either substituting the normalnucleobases G, A, T, C and U with derivatives having, for example,altered hydrogen bonding possibilities or by using LNAs that differ intheir backbone structure. The availability of such different LNAsfacilitates exquisite tuning of the affinity of modifiedoligonucleotides.

Another object of the present invention is to provide LNA modifiedoligonucleotides which are more resistant to nucleases than theirunmodified counterparts.

Another object of the present invention is to provide LNA modifiedoligonucleotides which can recruit RNAseH.

An additional object of the present invention is to provide LNAs thatcan act as substrates for DNA and RNA polymerases thereby allowing theanalogues to be either incorporated into a growing nucleic acid chain orto act as chain terminators.

A further object of the present invention is to provide LNAs that canact as therapeutic agents. Many examples of therapeutic nucleosideanalogues are known and similar derivatives of the nucleoside analoguesdisclosed herein can be synthesised using the procedures known from theliterature (E. De Clercq, J. Med. Chem. 1995, 38, 2491; P. Herdewijn andE. De Clercq: Classical Antiviral Agents and Design og New Antiviral

Agents. In: A Textbook of Drug Design and Development; Eds. P.Krogsgaard-Larsen, T. Liljefors and U. Madsen; Harwood AcademicPublishers, Amsterdam, 1996, p. 425; I. K. Larsen: Anticancer Agents.In:A Textbook of Drug Design and Development; Eds. P. Krogsgaard-Larsen, T.Liljefors and U. Madsen; Harwood Academic Publishers, Amsterdam, 1996,p. 460).

Double-stranded RNA has been demonstrated to posses anti-viral activityand tumour suppressing activity (Sharp et al., Eur. J. Biochem. 230(1):97-103, 1995, Lengyel-P. et al., Proc. Natl. Acad. Sci. U.S.A., 90(13):5893-5, 1993, and Laurent-Crawford et al., AIDS Res. Hum. Retroviruses,8(2): 285-90, 1992). It is likely that double stranded LNAs may mimicthe effect of therapeutically active double stranded RNAs andaccordingly such double stranded LNAs has a potential as therapeuticdrugs.

When used herein, the term “natural nucleic acid” refers to nucleicacids in the broadest sense, like for instance nucleic acids present inintact cells of any origin or vira or nucleic acids released from suchsources by chemical or physical means or nucleic acids derived from suchprimary sources by way of amplification. The natural nucleic acid may besingle, double or partly double stranded, and may be a relatively purespecies or a mixture of different nucleic acids. It may also be acomponent of a crude biological sample containing other nucleic acidsand other cellular components. On the other hand, the term “syntheticnucleic acids” refers to any nucleic acid produced by chemicalsynthesis.

The present invention also provides the use of LNA modifiedoligonucleotides in nucleic acid based therapeutic, diagnostics andmolecular biology. The LNA modified oligonucleotides can be used in thedetection, identification, capture, characterisation, quantification andfragmentation of natural or synthetic nucleic acids, and as blockingagents for translation and transcription in vivo and in vitro. In manycases it will be of interest to attach various molecules to LNA modifiedoligonucleotides. Such molecules may be attached to either end of theoligonucleotide or they may be attached at one or more internalpositions. Alternatively, they may be attached to the oligonucleotidevia spacers attached to the 5′ or 3′ end. Representative groups of suchmolecules are DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands. Generally all methods for labelling unmodified DNA and RNAoligonucleotides with these molecules can also be used to label LNAmodified oligonucleotides. Likewise, all methods used for detectinglabelled oligonucleotides generally apply to the corresponding labelled,LNA modified oligonucleotides.

Therapy

The term “strand displacement” relates to a process whereby anoligonucleotide binds to its complementary target sequence in a doublestranded DNA or RNA so as to displace the other strand from said targetstrand.

In an aspect of the present invention, LNA modified oligonucleotidescapable of performing “strand displacement” are exploited in thedevelopment of novel pharmaceutical drugs based on the “antigene”approach. In contrast to oligonucleotides capable of making triplehelices, such “strand displacement” oligonucleotides allow any sequencein a dsDNA to be targeted and at physiological ionic strength and pH.

The “strand displacing” oligonucleotides can also be used advantageouslyin the antisense approach in cases where the RNA target sequence isinaccessible due to intramolecular hydrogen bonds. Such intramolecularstructures may occur in mRNAs and can cause significant problems whenattempting to “shut down” the translation of the mRNA by the antisenseapproach.

Other classes of cellular RNAs, like for instance tRNAs, rRNAs snRNAsand scRNAs, contain intramolecular structures that are important fortheir function. These classes of highly structured RNAs do not encodeproteins but rather (in the form of RNA/protein particles) participatein a range of cellular functions such as mRNA splicing, polyadenylation,translation, editing, maintainance of chromosome end integrity, etc. Dueto their high degree of structure, that impairs or even prevent normaloligonucleotides from hybridising efficiently, these classes of RNAs hasso far not attracted interest as antisense targets.

The use of high affinity LNA monomers should facilitate the constructionof antisense probes of sufficient thermostability to hybridiseeffectively to such target RNAs. Therefore, in a preferred embodiment,LNA is used to confer sufficient affinity to the oligonucleotide toallow it to hybridise to these RNA classes thereby modulating thequalitative and/or quantitative function of the particles in which theRNAs are found.

In some cases it may be advantageous to down-regulate the expression ofa gene whereas in other cases it may be advantageous to activate it. Asshown by Møllegaard et al. (Møllegaard, N. E.; Buchardt, O.; Egholm, M.;Nielsen, P. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3892), oligomerscapable of “strand displacement” can function as RNA transcriptionalactivators. In an aspect of the present invention, the LNAs capable of“strand displacement” are used to activate genes of therapeuticinterest.

In chemotherapy of numerous viral infections and cancers, nucleosidesand nucleoside analogues have proven effective. LNA nucleosides arepotentially useful as such nucleoside based drugs.

Various types of double-stranded RNAs inhibit the growth of severaltypes of cancers. Duplexes involving one or more LNA oligonucleotide(s)are potentially useful as such double-stranded drugs.

The invention also concerns a pharmaceutical composition comprising apharmaceutically active LNA modified oligonucleotide or apharmaceutically active LNA monomer as defined above in combination witha pharmaceutically acceptable carrier.

Such compositions may be in a form adapted to oral, parenteral(intravenous, intraperitoneal), intramuscular, rectal, intranasal,dermal, vaginal, buccal, ocularly, or pulmonary administration,preferably in a form adapted to oral administration, and suchcompositions may be prepared in a manner well-known to the personskilled in the art, e.g. as generally described in “Remington'sPharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), MarkPublishing Company, Easton, Pa., U.S.A., 1985 and more recent editionsand in the monographs in the “Drugs and the Pharmaceutical Sciences”series, Marcel Dekker.

Diagnostics

Several diagnostic and molecular biology procedures have been developedthat utilise panels of different oligonucleotides to simultaneouslyanalyse a target nucleic acid for the presence of a plethora of possiblemutations. Typically, the oligonucleotide panels are immobilised in apredetermined pattern on a solid support such that the presence of aparticular mutation in the target nucleic acid can be revealed by theposition on the solid support where it hybridises. One importantprerequisite for the successful use of panels of differentoligonucleotides in the analysis of nucleic acids is that they are allspecific for their particular target sequence under the single appliedhybridisation condition. Since the affinity and specificity of standardoligonucleotides for their complementary target sequences depend heavilyon their sequence and size this criteria has been difficult to fulfil sofar.

In a preferred embodiment, therefore, LNAs are used as a means toincrease affinity and/or specificity of the probes and as a means toequalise the affinity of different oligonucleotides for theircomplementary sequences. As disclosed herein such affinity modulationcan be accomplished by, e.g., replacing selected nucleosides in theoligonucleotide with an LNA carrying a similar nucleobase. As furthershown herein, different classes of LNAs exhibit different affinities fortheir complementary nucleosides. For instance, the 2-3 bridged LNA withthe T-nucleobase exhibits less affinity for the A-nucleoside than thecorresponding 2-4 bridged LNA. Hence, the use of different classes ofLNAs offers an additional level for fine-tuning the affinity of aoligonucleotide.

In another preferred embodiment the high affinity and specificity of LNAmodified oligonucleotides is exploited in the sequence specific captureand purification of natural or synthetic nucleic acids. In one aspect,the natural or synthetic nucleic acids are contacted with the LNAmodified oligonucleotide immobilised on a solid surface. In this casehybridisation and capture occurs simultaneously. The captured nucleicacids may be, for instance, detected, characterised, quantified oramplified directly on the surface by a variety of methods well known inthe art or it may be released from the surface, before suchcharacterisation or amplification occurs, by subjecting the immobilised,modified oligonucleotide and captured nucleic acid to dehybridisingconditions, such as for example heat or by using buffers of low ionicstrength.

The solid support may be chosen from a wide range of polymer materialssuch as for instance CPG (controlled pore glass), polypropylene,polystyrene, polycarbonate or polyethylene and it may take a variety offorms such as for instance a tube, a micro-titer plate, a stick, a bead,a filter, etc. The LNA modified oligonucleotide may be immobilised tothe solid support via its 5′ or 3′ end (or via the terminus of linkersattached to the 5′ or 3′ end) by a variety of chemical or photochemicalmethods usually employed in the immobilisation of oligonucleotides or bynon-covalent coupling such as for instance via binding of a biotinylatedLNA modified oligonucleotide to immobilised streptavidin. One preferredmethod for immobilising LNA modified oligonucleotides on different solidsupports is photochemical using a photochemically active anthraquinonecovalently attached to the 5′ or 3′ end of the modified oligonucleotide(optionally via linkers) as described in (WO 96/31557). Thus, thepresent invention also provide a surface carrying an LNA modifiedoligonucleotide.

In another aspect the LNA modified oligonucleotide carries a ligandcovalently attached to either the 5′ or 3′ end. In this case the LNAmodified oligonucleotide is contacted with the natural or syntheticnucleic acids in solution whereafter the hybrids formed are capturedonto a solid support carrying molecules that can specifically bind theligand.

In still another aspect, LNA modified oligonucleotides capable ofperforming “strand displacement” are used in the capture of natural andsynthetic nucleic acids without prior denaturation. Such modifiedoligonucleotides are particularly useful in cases where the targetsequence is difficult or impossible to access by normal oligonucleotidesdue to the rapid formation of stable intramolecular structures. Examplesof nucleic acids containing such structures are rRNA, tRNA, snRNA andscRNA.

In another preferred embodiment, LNA modified oligonucleotides designedwith the purpose of high specificity are used as primers in thesequencing of nucleic acids and as primers in any of the several wellknown amplification reactions, such as the PCR reaction. As shownherein, the design of the LNA modified oligonucleotides determineswhether it will sustain a exponential or linear target amplification.The products of the amplification reaction can be analysed by a varietyof methods applicable to the analysis of amplification productsgenerated with normal DNA primers. In the particular case where the LNAmodified oligonucleotide primers are designed to sustain a linearamplification the resulting amplicons will carry single stranded endsthat can be targeted by complementary probes without denaturation. Suchends could for instance be used to capture amplicons by othercomplementary LNA modified oligonucleotides attached to a solid surface.

In another aspect, LNA modified oligos capable of “strand displacement”are used as primers in either linear or exponential amplificationreactions. The use of such oligos is expected to enhance overallamplicon yields by effectively competing with amplicon re-hybridisationin the later stages of the amplification reaction. Demers, et al. (Nucl.Acid Res. 1995, Vol 23, 3050-3055) discloses the use of high-affinity,non-extendible oligos as a means of increasing the overall yield of aPCR reaction. It is believed that the oligomers elicit these effect byinterfering with amplicon re-hybridisation in the later stages of thePCR reaction. It is expected that LNA modified oligos blocked at their3′ end will provide the same advantage. Blocking of the 3′ end can beachieved in numerous ways like for instance by exchanging the 3′hydroxyl group with hydrogen or phosphate. Such 3′ blocked LNA modifiedoligos can also be used to selectively amplify closely related nucleicacid sequences in a way similar to that described by Yu et al.(Biotechniques, 1997, 23, 714-716).

In recent years, novel classes of probes that can be used in for examplereal-time detection of amplicons generated by target amplificationreactions have been invented. One such class of probes have been termed“Molecular Beacons”. These probes are synthesised as partlyself-complementary oligonucleotides containing a fluorophor at one endand a quencher molecule at the other end. When free in solution theprobe folds up into a hairpin structure (guided by theself-complimentary regions) which positions the quencher in sufficientcloseness to the fluorophor to quench its fluorescent signal. Uponhybridisation to its target nucleic acid, the hairpin opens therebyseparating the fluorophor and quencher and giving off a fluorescentsignal.

Another class of probes have been termed “Taqman probes”. These probesalso contain a fluorophor and a quencher molecule. Contrary to theMolecular Beacons, however, the quenchers ability to quench thefluorescent signal from the fluorophor is maintained after hybridisationof the probe to its target sequence. Instead, the fluorescent signal isgenerated after hybridisation by physical detachment of either thequencher or fluorophor from the probe by the action of the 5′exonuxlease activity of a polymerase which has initiated synthesis froma primer located 5′ to the binding site of the Taqman probe.

High affinity for the target site is an important feature in both typesof probes and consequently such probes tends to be fairly large(typically 30 to 40 mers). As a result, significant problems areencountered in the production of high quality probes. In a preferredembodiment, therefore, LNA is used to improve production and subsequentperformance of Taqman probes and Molecular Beacons by reducing theirsize whilst retaining the required affinity.

In a further aspect, LNAs are used to construct new affinity pairs(either fully or partially modified oligonucleotides). The affinityconstants can easily be adjusted over a wide range and a vast number ofaffinity pairs can be designed and synthesised. One part of the affinitypair can be attached to the molecule of interest (e.g. proteins,amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, PNA,etc.) by standard methods, while the other part of the affinity pair canbe attached to e.g. a solid support such as beads, membranes,micro-titer plates, sticks, tubes, etc. The solid support may be chosenfrom a wide range of polymer materials such as for instancepolypropylene, polystyrene, polycarbonate or polyethylene. The affinitypairs may be used in selective isolation, purification, capture anddetection of a diversity of the target molecules mentioned above.

The principle of capturing an LNA-tagged molecule by ways of interactionwith another complementary LNA oligonucleotide (either fully orpartially modified) can be used to create an infinite number of novelaffinity pairs.

In another preferred embodiment the high affinity and specificity of LNAmodified oligonucleotides are exploited in the construction of probesuseful in in-situ hybridisation. For instance, LNA could be used toreduce the size of traditional DNA probes whilst maintaining therequired affinity thereby increasing the kinetics of the probe and itsability to penetrate the sample specimen. The ability of LNA modifiedoligonucleotides to “strand invade” double stranded nucleic acidstructures are also of considerable advantage in in-situ hybridisation,because it facilitates hybridisation without prior denaturation of thetarget DNA/RNA.

In another preferred embodiment, LNA modified oligonucleotides to beused in antisense therapeutics are designed with the dual purpose ofhigh affinity and ability to recruit RNAseH. This can be achieved by,for instance, having LNA segments flanking an unmodified central DNAsegment.

The present invention also provides a kit for the isolation,purification, amplification, detection, identification, quantification,or capture of natural or synthetic nucleic acids, where the kitcomprises a reaction body and one or more LNA modified oligonucleotides(oligomer) as defined herein. The LNA modified oligonucleotides arepreferably immobilised onto said reactions body.

The present invention also provides a kit for the isolation,purification, amplification, detection, identification, quantification,or capture of natural or synthetic nucleic acids, where the kitcomprises a reaction body and one or more LNAs as defined herein. TheLNAs are preferably immobilised onto said reactions body (e.g. by usingthe immobilising techniques described above).

For the kits according to the invention, the reaction body is preferablya solid support material, e.g. selected from borosilicate glass,soda-lime glass, polystyrene, polycarbonate, polypropylene,polyethylene, polyethyleneglycol terephthalate, polyvinylacetate,polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride,preferably polystyrene and polycarbonate. The reaction body may be inthe form of a specimen tube, a vial, a slide, a sheet, a film, a bead, apellet, a disc, a plate, a ring, a rod, a net, a filter, a tray, amicrotitre plate, a stick, or a multi-bladed stick.

The kits are typically accompanied by a written instruction sheetstating the optimal conditions for use of the kit.

The above-mentioned diagnostic and therapeutic aspects of the presentinvention have been illustrated with the following examples.

EXPERIMENTAL General

All reagents were obtained from commercial suppliers and were usedwithout further purification. After drying any organic phase usingNa₂SO₄, filtration was performed. The silica gel (0.040-0.063 mm) usedfor column chromatography was purchased from Merck. NMR spectra wererecorded at 300 MHz or 250 MHz for ¹H NMR and 62.9 MHz for ¹³C NMR andat 202.33 MHz for ³¹P NMR. 6-Values are in ppm relative totetramethylsilane as internal standard (¹H NMR and ¹³C NMR) and relativeto 85% H₃PO₄ as external standard (³¹P NMR). Assignments of NMR peaksare given according to standard nucleoside nomenclature. EI massspectra, FAB mass spectra and Plasma Desorption mass spectra wererecorded to gain information on the molecular weight of synthesisedcompounds. Oligonucleotide analogues were synthesised using thephosphoramidite methodology. Purification of 5′-O-DMT-ON or 5′-O-DMT-OFFoligonucleotide analogues was accomplished using disposable reversedphase chromatography cartridges or reversed phase HPLC when necessary.Matrix-assisted laser desorption mass spectra were obtained to verifythe molecular weight and monomer composition of representativeoligonucleotide samples. Capillary gel electrophoresis was performed toverify the purity of representive oligonucleotide samples.

The specific descriptions below are accompanied by FIGS. 2-41 and Tables1-10. Unless otherwise stated in the following examples, “LNA”designates the 2′-4′-bridged variant illustrated with the forumula Z inFIG. 2.

Preparation of LNA Monomers Example 1

3-C-Allyl-1,2-O-isopropylidene-α-D-ribofuranose (OA). Method 1: Asolution of5-O-t-butyldimethylsilyl-1,2-O-isopropylidene-α-D-ribofuran-3-ulose (Y.Yoshimura, T. Sano, A. Matsuda and T. Ueda, Chem. Pharm. Bull., 1988,36, 162) (17.8 g, 58.9 mmol) in anhydrous THF (980 cm³) was stirred at0° C. and 1 M allylmagnesium bromide in anhydrous ether (130 cm³, 130mmol) was added dropwise. After stiffing for 2 h, a saturated aqueoussolution of ammonium chloride (800 cm³) was added and the mixture wasextracted with dichloromethane (3×400 cm³). The organic phase was washedwith brine (3×450 cm³) and dried (Na₂SO₄). The solvent was removed underreduced pressure and the residue was dissolved in anhydrous THF (700cm³). A 1.1 M solution of tetrabutylammonium fluoride in THF (54.4 cm³,59.8 mmol) was added and the mixture was stirred at room temperature for1 h and evaporated to dryness. The residue was dissolved indichloromethane (1700 cm³) and was washed with a saturated aqueoussolution of sodium hydrogencarbonate (3×500 cm³) and dried (Na₂SO₄). Thesolvent was removed under reduced pressure and the residue was purifiedby silica gel column chromatography using dichloromethane/methanol(98:2, v/v) as eluent to give furanose 0A as a white solid material(9.42 g, 69%). Method 2: Furanose 0A was analogously synthesised from5-O-t-butyldiphenylsilyl-1,2-O-isopropylidene-α-D-ribofuran-3-ulose (T.F. Tam and B. Fraser-Reid, J. Chem. Soc., Chem. Commun., 1980, 556) (9.5g, 22.2 mmol) using: anhydrous THF (425 cm³); a 1 M solution ofallylmagnesium bromide in anhydrous ether (130 cm³, 130 mmol); asaturated aqueous solution of ammonium chloride (490 cm³); ether forextraction (350+2×160 cm³); brine (2×160 cm³); a 1.1 M solution oftetrabutylammonium fluoride in THF (22.3 cm³, 24.6 mmol); anhydrous THF(400 cm³); dichloromethane (1400 cm³); a saturated aqueous solution ofsodium hydrogencarbonate (3×500 cm³); brine (500 cm³) and (Na₂SO₄).δ_(H) ((CD₃)₂SO) 5.84 (1 H, m, 2′-H), 5.65 (1H, d, J 3.8, 1-H), 5.12(1H, d, J 6.1, 3′-H_(a)), 5.06 (1H, br s, 3′-H_(b)), 4.76 (1H, s, 3-OH),4.64 (1H, t, J 5.4, 5-OH), 4.16 (1H, d, J 3.8, 2-H), 3.84 (1H, dd, J2.2, 8.1, 4-H), 3.56 (1H, ddd, J 2.3, 5.6, 11.8, 5-H_(a)), 3.42 (1H, m,5-H_(b)), 2.16 (1H, dd, J 6.1, 14.3, 1′-H_(a)), 1.98 (1H, dd, J 8.2,14.3, 1′-H_(b)), 1.46 (3H, s, CH₃), 1.25 (3H, s, CH₃). δ_(C) (CDCl₃)133.5 (C-2′), 117.9 (C-3′), 110.8 (C(CH₃)₂), 102.9 (C-1), 82.6, 81.0,77.7 (C-2, C-3, C-4), 59.4 (C-5), 36.4 (C-1′), 26.4, 26.3 (CH₃) (Found:C, 57.4; H, 8.0; C₁₁H₁₈O₅ requires C, 57.4; H, 7.9%).

Example 2

3-C-Allyl-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose (0B). A60% suspension of sodium hydride (4.9 g, 123 mmol) in anhydrous DMF (100cm³) was stirred at 0° C. and a solution of furanose 0A (9.42 g, 40.9mmol) in anhydrous DMF (65 cm³) was added dropwise over 45 min. Thesolution was stirred for 1 h at 50° C. and cooled to 0° C. A mixture ofbenzyl bromide (14.5 cm³, 121 mmol) and anhydrous DMF (14.5 cm³) wasadded dropwise and the mixture was stirred at room temperature for 18 h.The reaction mixture was evaporated to dryness and a solution of theresidue in dichloromethane (700 cm³) was washed with a saturated aqueoussolution of sodium hydrogencarbonate (2×450 cm³) and dried (Na₂SO₄). Thesolvent was removed under reduced pressure and the residue was purifiedby silica gel column chromatography using petroleum ether/ethylacetate(9:1, v/v) as eluent to give compound 0B as an oil (14.5 g, 86%). δ_(H)(CDCl₃) 7.39-7.21 (10H, m, Bn), 5.92 (1H, m, 2′-H), 5.71 (1H, d, J 3.8,1-H), 5.17-5.09 (2H, m, 3′-H_(a), 3′-H_(b)), 4.67 (2H, m, Bn), 4.60 (1H,d, J 12.2, Bn), 4.52 (1H, d, J 12.1, Bn), 4.43 (1H, m, 4-H), 4.42 (1H,d, J 3.8, 2-H), 3.73 (1H, dd, J 3.2, 10.8, 5-H_(a)), 3.66 (1H, dd, J7.4, 10.8, 5-H_(b)), 2.50 (1H, dd, J 7.7, 14.9, 1′-H_(a)), 2.39 (1H, dd,J 6.5, 14.9, 1′-H_(b)), 1.60 (3H, s, CH₃), 1.34 (3H, s, CH₃). δ_(C)(CDCl₃) 138.7, 138.1 (Bn), 132.6 (C-2′), 128.3, 128.2, 127.7, 127.5,127.4, 127.4 (Bn), 118.5 (C-3′), 112.6 (C(CH₃)₂), 104.1 (C-1), 86.5,82.1, 80.4 (C-2, C-3, C-4), 73.4, 68.6 (Bn), 67.0 (C-5), 35.8 (C-1′),26.8, 26.6 (CH₃). FAB-MS m/z 433 [M+Na]⁺ (Found: C, 73.4; H, 7.4;C₂₅H₃₀O₅ requires C, 73.2; H, 7.4%).

Example 3

3-C-Allyl-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribofuranose (0C). Asolution of furanose 0B (12.42 g, 30.3 mmol) in 80% aqueous acetic acid(150 cm³) was stirred at 90° C. for 3 h. The solvent was removed underreduced pressure and the residue was coevaporated with ethanol (3×75cm³), toluene (3×75 cm³) and anhydrous pyridine (2×75 cm³) andredissolved in anhydrous pyridine (60 cm³). Acetic anhydride (46 cm³)was added and the solution was stirred at room temperature for 48 h. Amixture of ice and water (300 cm³) was added and the resulting mixturewas extracted with dichloromethane (2×300 cm³). The combined organicphase was washed with a saturated aqueous solution of sodiumhydrogencarbonate (3×200 cm³) and dried (Na₂SO₄). The solvent wasevaporated and the residue was purified using silica gel columnchromatography with petroleum ether/ethyl acetate (4:1, v/v) as eluentto give the anomeric mixture 0C (β:α˜2:1) as an oil (13.3 g, 97%). δ_(C)(CDCl₃) 169.7, 169.6 (C═O), 138.7, 138.4, 137.7, 137.6 (Bn), 132.4,132.2 (C-2′), 128.4 128.4, 128.2, 128.2, 127.8, 127.7, 127.7, 127.6,127.3, 127.3, 126.9, 126.8 (Bn), 118.5 (C-3′), 99.4, 93.5 (C-1), 84.8,83.7, 83.2, 82.0, 79.1, 75.5 (C-2, C-3, C-4), 73.7, 73.5, 69.3, 68.7(Bn), 66.1 (C-5), 35.5, 34.9 (C-1), 21.1, 21.0, 20.7, 20.6 (CH₃) (Found:C, 68.7; H, 6.7; C₂₆H₃₀O₇ requires C, 68.8; H, 6.6%).

Example 4

1-(2-O-Acetyl-3-C-allyl-3,5-di-O-benzyl-β-D-ribofuranosyl)thymine (1).To a stirred solution of the anomeric mixture 0C (β:α˜2:1, 11.8 g, 26.0mmol) (P. Nielsen, H. M. Pfundheller and J. Wengel, Chem. Commun., 1997,825; P. Nielsen, H. M. Pfundheller, C. E. Olsen and J. Wengel, J. Chem.Soc., Perkin Trans. 1, 1997, in the press) and thymine (6.55 g, 52.0mmol) in anhydrous acetonitrile (250 cm³) was addedN,O-bis(trimethylsilyl)acetamide (44.9 cm³, 182 mmol). The reactionmixture was stirred at reflux for 1 h and cooled to 0° C. Trimethylsilyltriflate (8.00 cm³, 44.0 mmol) was added dropwise and the solution wasstirred at room temperature for 12 h. An ice-cold saturated aqueoussolution of sodium hydrogencarbonate (270 cm³) was added and the mixturewas extracted with dichloromethane (3×125 cm³). The organic phase waswashed with saturated aqueous solutions of sodium hydrogencarbonate(2×125 cm³) and brine (2×125 cm³) and dried (Na₂SO₄). The solvent wasremoved under reduced pressure and the residue was purified by silicagel column chromatography using dichloromethane/methanol (98:2, v/v) aseluent to give nucleoside 1 as a white solid material (11.6 g, 86%).δ_(H) (CDCl₃) 8.64 (1H, br s, NH), 7.75 (1H, d, J 1.1, 6-H), 7.41-7.25(10H, m, Bn), 6.43 (1H, d, J 8.2, 1′-H), 5.88 (1H, m, 2″-H), 5.66 (1H,d, J 8.2, 2′-H), 5.12 (1H, s, 3″-H_(a)), 5.07 (1H, dd, J 1.5, 8.5,3″-H_(b)), 4.85 (1H, d, J 11.2, Bn), 4.64 (2 H, s, Bn), 4.63 (1H, d, J11.2, Bn), 4.33 (1H, br s, 4′-H), 3.81 (1H, dd, J 2.7, 11.1, 5′-H_(a)),3.65 (1H, m, 5′-H_(b)), 2.81-2.65 (2H, m, 1″-H_(a), 1″-H_(b)), 2.08 (3H,s, COCH₃), 1.52 (3 H, d, J 0.8, CH₃). (CDCl₃) 170.1 (C═O), 163.6 (C-4),150.9 (C-2), 138.1, 136.6 (Bn), 136.0 (C-6), 131.6 (C-2″), 128.8, 128.4,128.3, 127.6, 127.5, 127.1 (Bn), 118.5 (C-3″), 111.1 (C-5), 84.2, 83.4,83.1, 77.4 (C-1′, C-2′, C-3′, C-4′), 73.6, 69.2 (Bn), 65.6 (C-5′), 33.7(C-1″), 20.8 (COCH₃), 11.9 (CH₃) (Found: C, 66.8; H, 6.3; N, 5.1.C₂₉H₃₂N₂O₇ requires C, 66.9; H, 6.2; N, 5.4%).

Example 5

1-(3-C-Allyl-3,5-di-O-benzyl-β-D-ribofuranosyl)thymine (2). To a stirredsolution of nucleoside 1 (11.6 g, 22.3 mmol) in methanol (110 cm³) wasadded sodium methoxide (3.03 g, 55.5 mmol). The reaction mixture wasstirred at room temperature for 16 h and neutralised with dilutehydrochloric acid. The solvent was partly evaporated and the residue wasdissolved in dichloromethane (2×400 cm³). The organic phase was washedwith a saturated aqueous solution of sodium hydrogencarbonate (3×250cm³) and dried (Na₂SO₄). The solvent was removed under reduced pressureto give 2 as a white solid material (10.1 g, 95%). δ_(H) (CDCl₃) 8.77(1H, br s, NH), 7.58 (1H, d, J 1.2, 6-H), 7.41-7.25 (10H, m, Bn), 6.14(1H, m, 2″-H), 6.12 (1H, d, J 7.8, 1′-H), 5.23 (1H, m, 3″-H_(a)), 5.17(1H, br s, 3″-H_(b)), 4.68 (1H, d, J 10.8, Bn), 4.59 (2H, s, Bn), 4.55(1H, d, J 10.9, Bn), 4.39 (1H, br s, 4′-H), 4.26 (1H, dd J 7.8, 10.7,2′-H), 3.84 (1H, dd, J 3.1, 11.0, 5′-H_(a)), 3.58 (1H, dd, J 1.4, 11.0,5′-H_(b)), 3.04 (1H, d, J 10.8, 2′-OH), 2.82-2.78 (2 H, m, 1″-H_(a),1″-H_(b)), 1.51 (3H, d, J1.0, CH₃). δ_(C) (CDCl₃) 163.5 (C-4), 151.1(C-2), 137.3, 136.7 (Bn), 136.0 (C-6), 132.1 (C-2″), 128.8, 128.5,128.3, 127.9, 127.6 (Bn), 118.4 (C-3″), 111.1 (C-5), 87.4, 82.6, 81.1,79.3 (C-1′, C-2′, C-3′, C-4′), 73.7, 69.8 (Bn), 64.7 (C-5′), 35.1(C-1″), 11.9 (CH₃). (Found: C, 67.8; H, 6.1; N, 5.5. C₂₇H₃₀N₂O₆ requiresC, 67.8; H, 6.3; N, 5.9%).

Example 6

1-(3-C-Allyl-3,5-di-O-benzyl-2-O-methanesulfonyl-β-D-ribofuranosyl)thymine(3). To a stirred solution of nucleoside 2 (3.50 g, 7.31 mmol) inanhydrous pyridine (23 cm³) at 0° C. was added methanesulphonyl chloride(1.69 cm³, 21.89 mmol). The reaction mixture was stirred for 1 h at roomtemperature, water (100 cm³) was added and extraction was performedusing dichloromethane (3×150 cm³). The organic phase was washed with asaturated aqueous solution of sodium hydrogencarbonate (3×200 cm³) anddried (Na₂SO₄). The solvent was removed under reduced pressure and theresidue purified by silica gel column chromatography usingdichloromethane/methanol (99:1) as eluent to give 3 as a white solidmaterial (3.64 g, 89%). δ_(H) (CDCl₃) 8.95 (1H, br s, NH), 7.71 (1H, d,J 1.1, 6-H), 7.39-7.25 (10H, m, Bn), 6.52 (1H, d, J 8.0, 1′-H), 5.90(1H, m, 2″-H), 5.34 (1H, d, J 7.9, 2′-H), 5.20-5.09 (2H, m, 3″-H_(a),3″-H_(b)), 4.91 (1H, d, J 11.2, Bn), 4.68 (1H, d, J 11.3, Bn), 4.64 (2H,s, Bn), 4.33 (1H, br s, 4′-H), 3.81 (1H, dd, J 2.5, 11.1, 5′-H_(a)),3.73 (1H, dd, J 1.1, 11.1, 5′-H_(b)), 3.08 (1H, dd, J 5.5, 5.7,1″-H_(a)), 2.99 (3H, s, CH₃), 2.68 (1H, m, 1″-H_(b)), 1.51 (3H, d, J0.8, CH₃). δ_(C) (CDCl₃) 163.4 (C-4), 150.8 (C-2), 137.9, 136.3 (Bn),135.5 (C-6), 131.0 (C-2″), 128.8, 128.3, 127.5, 127.2 (Bn), 119.3(C-3″), 111.6 (C-5), 84.1, 83.6, 82.4, 82.2 (C-1′, C-2′, C-3′, C-4′),73.7, 68.9 (Bn), 66.2 (C-5′), 38.7 (CH₃), 33.0 (C-1″), 11.9 (CH₃)(Found: C, 60.5; H, 5.8; N, 4.9. C₂₈H₃₂N₂O₈S requires C, 60.4; H, 5.8;N, 5.0%).

Example 7

1-(3-C-Allyl-3,5-di-O-benzyl-β-D-arabinofuranosyl)thymine (4). Asolution of nucleoside 3 (3.59 g, 6.45 mmol) in ethanol (72 cm³), water(72 cm³) and 1 M aqueous sodium hydroxide (20.6 cm³) was stirred underreflux for 18 h. After neutralisation with dilute hydrochloric acid, thesolvent was removed under reduced pressure and the residue was dissolvedin dichloromethane (3×150 cm³). The organic phase was washed with asaturated aqueous solution of sodium hydrogencarbonate (3×200 cm³) anddried (Na₂SO₄). The solvent was removed under reduced pressure and theresidue was purified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give 4 as a whitesolid material (2.32 g, 74%). δ_(H) (CDCl₃) 7.60 (1H, d, J 1.2, 6-H),7.50-7.23 (10H, m, Bn), 6.22 (1H, d, J 2.9, 1′-H), 5.80 (1H, m, 2″-H),5.15-5.08 (2 H, m, 3″-H_(a), 3″-H_(b)), 4.86-4.33 (6H, m, 2×Bn, 2′-H,4′-H), 3.82-3.71 (2H, m, 5′-H_(a), 5′-H_(b)), 2.72 (1H, m, 1″-H_(a)),2.52 (1H, dd, J 7.6, 16.1, 1″-H_(b)), 1.70 (3H, d, J 0.9, CH₃). (CDCl₃)165.1 (C-4), 150.4 (C-2), 138.4, 136.8 (Bn), 137.7 (C-6), 132.3 (C-2″),128.77 128.4, 128.3, 128.0, 127.9, 127.6 (Bn), 118.5, (C-3″), 107.8(C-5), 88.0, 87.8, 83.7 (C-1′, C-3′, C-4′), 73.7, 72.9, 69.4 (Bn, C-2′),64.7 (C-5′), 31.1 (C-1″), 12.4 (CH₃) (Found: C, 67.5; H, 6.3; N, 5.3.C₂₇H₃₀N₂O₆, 0.25H₂O requires C, 67.1; H, 6.4; N, 5.8%).

Example 8

1-(3,5-Di-O-benzyl-3-C-(2-hydroxyethyl)-β-D-arabinofuranosyl)thymine(5). To a stirred solution of nucleoside 4 (2.26 g, 4.68 mmol) in THF(12 cm³) and water (12 cm³) was added sodium periodate (3.04 g, 14.2mmol) and a 2.5% solution of osmium tetraoxide in tert-butanol (w/w,0.603 cm³, 40 μmol). The solution was stirred at room temperature for 45min. Water (25 cm³) was added and the solution was extracted withdichloromethane (2×50 cm³). The organic phase was washed with asaturated aqueous solution of sodium hydrogencarbonate (3×30 cm³) anddried (Na₂SO₄). The solvent was removed under reduced pressure and theresidue was redissolved in THF (12 cm³) and water (12 cm³). The mixturewas stirred at room temperature and sodium boronhydride (182 mg, 4.71mmol) was added. After stirring for 1.5 h, water (25 cm³) was added andthe solution was extracted with dichloromethane (2×50 cm³). The organicphase was washed with a saturated aqueous solution of sodiumhydrogencarbonate (3×30 cm³) and dried (Na₂SO₄). The solvent was removedunder reduced pressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (98:2, v/v) as eluent togive 5 as a white solid material (1.13 g, 49%). δ_(H) (CDCl₃) 9.29 (1H,br s, NH), 7.47 (1H, d, J 1.1, 6-H), 7.38-7.25 (10H, m, Bn), 6.22 (1H,d, J 3.4, 1′-H), 4.62 (2H, s, Bn), 4.60 (1H, m, 4′-H), 4.46 (2H, s, Bn),4.35 (1H, dd, J 3.4, 7.5, 2′-H), 3.83-3.73 (4H, m, 2×5′-H, 2×2″-H), 2.67(1H, br s, OH), 2.07-2.01 (2H, m, 2×1″-H), 1.77 (3H, d, J 0.5, CH₃).δ_(C) (CDCl₃) 164.3 (C-4), 150.3 (C-2), 137.6, 137.4 (Bn, C-6), 136.7(Bn), 128.6, 128.4, 128.2, 127.8, 127.6, 127.3, 127.1 (Bn), 108.4 (C-5),88.0, 87.7, 81.6, 74.7 (C-1′, C-2′, C-3′, C-4′), 73.7, 69.6 (Bn), 64.6(C-5′), 57.7 (C-2″), 28.6 (C-1″), 12.4 (CH₃). FAB-MS m/z 483 [M+H]⁺, 505[M+Na]⁺ (Found: C, 63.6; H, 6.2; N, 5.4. C₂₆H₃₀N₂O₇,0.5H₂O requires C,63.5; H, 6.4; N, 5.7%).

Example 9

(1S,5R,6R,8R)-5-Hydroxy-6-(hydroxymethyl)-8-(thymin-1-yl)-2,7-dioxabicyclo-[3.3.0]octane(6). A solution of nucleoside 5 (1.08 g, 2.20 mmol) in anhydrouspyridine (5.0 cm³) was stirred at 0° C. and a solution ofp-toluenesulphonyl chloride (462 mg, 2.47 mmol) in anhydrous pyridine(2.0 cm³) was added dropwise. After stirring at room temperature for 20h and addition of a mixture of water and ice (70 cm³), extraction wasperformed with dichloromethane (2×75 cm³). The organic phase was washedwith a saturated aqueous solution of sodium hydrogencarbonate (3×50 cm³)and dried (Na₂SO₄). The solvent was removed under reduced pressure andthe residue was purified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give an intermediatewhich after evaporation was dissolved in anhydrous DMF (4.0 cm³). Thesolution was added dropwise to a stirred suspension of 60% sodiumhydride (203 mg, 4.94 mmol) in anhydrous DMF (4.0 cm³) at 0° C. Themixture was stirred for 18 h and water (20 cm³) was added. Afterneutralisation with hydrochloric acid, dichloromethane (75 cm³) wasadded. The organic phase was washed with a saturated aqueous solution ofsodium hydrogencarbonate (3×50 cm³) and dried (Na₂SO₄). The solvent wasremoved under reduced pressure and the residue was purified by silicagel column chromatography using dichloromethane/methanol (98:2, v/v) aseluent to give a white solid material material (858 mg). A solution ofthis white solid material (846 mg, 1.80 mmol) in ethanol (10.0 cm³) wasstirred at room temperature and 20% palladium hydroxide over carbon (400mg) was added. The mixture was degassed with argon and placed in ahydrogen atmosphere. After stirring for 2 h the mixture was directlypurified by silica gel column chromatography usingdichloromethane/methanol (97:3, v/v) as eluent to give 6 as a whitesolid material (444 mg, 82%). δ_(H) ((CD₃)₂SO) 11.3 (1H, br s, NH), 7.36(1H, d, J 1.1, 6-H), 5.80 (1H, d, J 4.3, 1′-H), 5.61 (1H, s, OH), 4.86(1H, m, 5′-H_(a)), 3.89 (1H, d, J 4.2, 2′-H), 3.85 (1H, m, 2″-H_(a)),3.83-3.64 (3H, m, 4′-H, 5′-H_(b), 2″-H_(b)), 2.14 (1H, m, 1″-H_(a)),1.81 (1H, m, 1″-H_(b)), 1.78 (3H, d, J 1.0, CH₃). δ_(C) (CD₃OD) 166.7(C-4), 152.2 (C-2), 139.7 (C-6), 110.1 (C-5), 89.4, 89.1, 85.5, 85.2(C-1′, C-2′, C-3′, C4′), 71.4 (C-2″), 61.6 (C-5′), 37.0 (C-1″), 12.7(CH₃) (Found: C, 47.4; H, 5.7; N, 9.0. C₁₂H₁₆N₂O₆,H₂O requires C, 47.7;H, 6.0; N, 9.3%).

Example 10

(1S,5R,6R,8R)-6-(4,4′-Dimethoxytrityloxymethyl)-5-hydroxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]nonane(7). A solution of nucleoside 6 (310 mg, 1.09 mmol) in anhydrouspyridine (2.5 cm³) was stirred at room temperature and4,4′-dimethoxytrityl chloride (593 mg, 1.83 mmol) was added. Afterstirring for 3 h, additional 4,4′-dimethoxytrityl chloride (100 mg,0.310 mmol) was added. After stirring for another 2 h, methanol (0.5cm³) was added and the mixture was evaporated. The residue was dissolvedin dichloromethane (5 cm³) and washed with an aqueous saturated solutionof sodium hydrogencarbonate (3×5 cm³). The organic phase was dried(Na₂SO₄) and evaporated under reduced pressure. The residue was purifiedby silica gel column chromatography with dichloromethane/methanol (99:1,v/v) as eluent to give 7 as a white solid material (618 mg, 97%). δ_(H)(CDCl₃) 9.04 (1H, br s, NH), 7.47-7.16 (10H, m, 6-H, DMT), 6.86-6.82(4H, m, DMT), 6.06 (1H, d, J 4.1, 1′-H), 4.35 (1H, d, J 4.1, 2′-H), 4.03(1H, m, 4′-H), 3.89 (1H, m, 2″-H_(a)), 3.79 (6H, s, 2×OCH₃), 3.61 (1H,m, 5′-H_(a)), 3.32-3.26 (2H, m, 5′-H_(b), 2″-H_(b)), 1.94-1.69 (2H, m,1″-H_(a), 1″-H_(b),), 1.89 (3H, s, CH₃). δ_(C) (CDCl₃) 163.4 (C-4),158.6 (DMT), 150.1 (C-2), 144.3 (DMT), 137.2 (C-6), 135.6, 135.3, 129.9,129.9, 128.9, 128.1, 127.9, 126.9, 125.2, 113.2 (DMT), 109.3 (C-5),88.7, 87.3, 86.9, 83.5, 81.0 (DMT, C-1′, C-2′, C-3′, C4′), 69.7 (C-2″),62.1 (C-5′), 55.1 (OCH₃), 36.5 (C-1″), 12.5 (CH₃).

Example 11

(1S,5R,6R,8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-6-(4,4′-dimethoxytrityloxymethyl)-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]nonane(8). A solution of nucleoside 7 (436 mg, 0.743 mmol) in anhydrousdichloromethane (2.2 cm³) and diisopropylethylamine (0.62 cm³) wasstirred at room temperature and 2-cyamoethylN,N-diisopropylphosphoramidochloridite (0.33 cm³, 1.46 mmol) was added.After stirring for 1.5 h, methanol (0.4 cm³) and ethyl acetate (5 cm³)were added and the mixture was washed with aqueous saturated solutionsof sodium hydrogencarbonate (3×5 cm³) and brine (3×5 cm³). The organicphase was dried (Na₂SO₄) and evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography usingdichloromethane/triethylamine (97:3, v/v) as eluent, the solvents wereevaporated to give an oil which was dissolved in toluene (1 cm³) andprecipitation from hexane at −30° C. yielded 8 as a solid white material(517 mg, 88%). δ_(P) (CDCl₃) 142.0, 141.9.

Example 12

1-(3,5-Di-O-benzyl-3-C-(2-hydroxyethyl)-β-D-ribofuranosyl)thymine (9).To a stirred solution of nucleoside 2 (1.00 g, 2.09 mmol) in THF (5.4cm³) and water (5.4 cm³) was added sodium periodate (1.34 g, 6.27 mmol)and a 2.5% solution of osmium tetraoxide in tert-butanol (w/w, 0.265cm³, 19 μmol). The solution was stirred at room temperature for 45 min.Water (25 cm³) was added and the solution was extracted withdichloromethane (2×50 cm³). The organic phase was washed with asaturated aqueous solution of sodium hydrogencarbonate (3×30 cm³) anddried (Na₂SO₄). The solvent was removed under reduced pressure and theresidue was redissolved in THF (5.4 cm³) and water (5.4 cm³). Themixture was stirred at room temperature and sodium boronhydride (79 mg,2.08 mmol) was added. After stirring for 1.5 h, water (25 cm³) was addedand the solution was extracted with dichloromethane (2×50 cm³). Theorganic phase was washed with a saturated aqueous solution of sodiumhydrogencarbonate (3×30 cm³) and dried (Na₂SO₄). The solvent was removedunder reduced pressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (98:2, v/v) as eluent togive nucleoside 9 as a white solid material (488 mg, 48%). δ_(H) (CDCl₃)9.14 (1 H, br s, NH), 7.60 (1H, d, J 1.1, 6-H), 7.40-7.22 (10H, m, Bn),6.25 (1H, d, J 7.7, 1′-H), 4.59 (1H, d, J 7.1 Bn), 4.49 (1H, d, J 7.1Bn), 4.39-3.30 (m, 8H, 4′-H, 2′-H, Bn, 5′-H_(a), 5′-H_(b), 2″-H_(a),2″-H_(b)), 2.23-2.00 (2H, m, 1″-H_(a), 1″-H_(b)), 1.49 (3H, d, J 0.7,CH₃). δ_(C) (CDCl₃) 163.5 (C-4), 151.2 (C-2), 137.1, 136.5 (Bn), 135.7(C-6), 128.7, 128.5, 128.2, 127.8, 127.6, 127.2 (Bn), 111.3 (C-5), 87.0,82.7, 81.1, 78.3 (C-1′, C-2′, C-3′, C-4′), 73.7, 69.6 (Bn), 64.4 (C-5′),57.0 (C-2″), 32.4 (C-1″), 11.8 (CH₃) (Found: C, 63.9; H, 6.3; N, 5.4.C₂₆H₃₀N₂O₇,0.25H₂O requires C, 64.1; H, 6.3; N, 5.75%).

Example 13

1-[3-C-(2-O-t-Butyldimethylsilyloxyethyl)-3,5-di-O-benzyl-β-D-ribofuranosyl]-thymine(10). A mixture of nucleoside 9 (1.80 g, 3.4 mmol) andt-butyldimethylsilyl chloride (0.585 g, 3.9 mmol) was dissolved inanhydrous pyridine (20 cm³). After 2 h at room temperature the reactionmixture was evaporated to dryness, twice co-evaporated with toluene(2×10 cm³) and re-dissolved in dichloromethane (150 cm³). The solutionwas washed with a saturated aqueous solution of sodium hydrocarbonate(2×50 cm³) and evaporated to give a foam. This material was purified bypreparative silica-gel HPLC using gradient elution (0-3% methanol indichloromethane, v/v) to give nucleoside 10 as a white solid material(1.86 g, 92%). δ_(H) (CDCl₃) 7.61 (1H, d, J 1.1, 6-H), 7.35-7.20 (10H,m, Bn), 6.27 (1H, d, J7.9, 1′-H), 4.65-4.40 (4H, m, Bn, 2′-H), 4.37 (1H,s, Bn), 4.28 (1H, t, J 7.9, 4′-H), 4.35-3.55 (4H, m, 2″-H_(a), 2″-H,5′-H_(a), 5′-H_(b)), 2.30-2.05 (2H, m, 1″-H_(a), 1″-H_(b)), 1.46 (3H, s,5-CH₃), 0.90 (9H, m, CH₃—C—Si), 0.08 (6H, m, CH₃—Si). δ_(C) (CDCl₃)163.6 (C-6), 151.0 (C-2), 137.5, 136.6, 135.8 (C-5, Bn), 128.3, 128.1,127.8, 127.2, 127.1, 126.8, 126.7 (Bn), 110.7 (C-4), 86.8, 82.5, 81.6,78.3 (C-1′, C-2′, C-3′, C-4′), 73.3, 69.8 (Bn), 64.46 (C-5′), 58.2(C-2″), 32.9 (C-1″), 25.6, 25.4, 17.9, −3.9, −5.7 (TBDMS), 11.6 (CH₃).FAB⁺-MS: m/z 597.19 [M+H]⁺, 619.18 [M+Na]⁺ (Found: C, 64.2; H, 7.4; N,4.2; C₃₂H₄₄O₇N₂Si requires C, 64.4; H, 7.4; N, 4.7%).

Example 14

1-[3-C-(2-t-Butyldimethylsilyloxyethyl)-3,5-di-O-benzyl-β-D-erythro-pentofuran-2-ulosyl]thymine(11). A mixture nucleoside 10 (2.14 g, 3.59 mmol), 1.48 g (3.95 mmol) ofpyridinium dichromate (1.48 g, 3.95) and activated 3 Å molecular sievepowder (4 g) was suspended in anhydrous dichloromethane (80 cm³). Aftercooling the mixture to −10° C., acetic anhydride (10 cm³, 98 mmol) wasadded dropwise under vigorous stiffing. The suspension was allowed towarm to room temperature and stirring was continued for 1.5 h whereuponthe reaction was quenched by addition of triethylamine (20 cm³). Themixture was diluted with dichloromethane to 300 cm³ and was washed withwater (2×200 cm³). The organic phase was evaporated, and the residuepurified by flash silica-gel chromatography using a gradient of 1.0,1.2, 1.3, 1.4, 1.5% methanol in dichloromethane (v/v, total volume 250cm³ each) to give nucleoside 11 (1.89 g, 84.4%) as a white solidmaterial. δ_(H) (CDCl₃) 7.35-7.20 (11H, m, Bn, 6-H), 6.40 (1H, s, 1′-H),4.57 (1H, s, Bn), 4.52 (1H, s, Bn), 4.46 (1H, d, J 11.0, Bn), 4.29 (1H,d, J 11.0, Bn), 4.07 (1H, dd, J′ 0.5, 2.2, 4′-H), 3.95-3.70 (4H, m,2″-H_(a), 2″-H_(b), 5′-H_(a), 5′-H_(b)), 2.05 (1H, m, 1″-H_(a)), 2.42(1H, m, 1″-H_(b)), 1.42 (3H, d, J 1.1, 5-CH₃), 0.86 (9H, s, CH₃—C—Si),0.01 (6H, s, CH₃—Si). δ_(C) (CDCl₃) 202.6 (C-2′), 163.7 (C-4), 151.2(C-2), 137.7, 136.6, 136.5 (Bn, C-6), 128.7, 128.5, 128.2, 128.1, 127.7,126.4, 126.3 (Bn), 110.9 (C-5), 84.5, 81.3, 80.2 (C-1′, C-3′, C-4′),73.6, 70.4 (Bn), 66.0 (C-5′), 57.6 (C-2″), 27.3 (C-1″), 25.9, 25.7,18.2, −5.8, −5.9 (TBDMS), 11.7 (CH₃). FAB-MS m/z 595.14 [M+H]⁺ (Found:C, 64.1; H, 6.9; N, 4.5; C₃₂H₄₂O₇N₂Si requires C, 64.6; H, 7.1; N,4.7%).

Example 15

(1S,5R,6R,8R)-1-Hydroxy-5-benzyloxy-6-benzyloxymethyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane(12). Compound II (1.80 g, 30.3 mmol) was dissolved in 0.5% HCl inmethanol (w/w, 20 cm³) and the mixture was stirred for 30 min at roomtemperature. After evaporation to dryness, the residue was dissolved indichloromethane (100 cm³) and washed with a saturated aqueous solutionof sodium hydrogencarbonate (2×40 cm³). The organic phase was evaporatedand the residue was purified by flash silica-gel chromatography elutingwith 2% methanol in dichloromethane (v/v) to yield nucleoside 12 (1.35g, 93.5%) as a white solid material. δ_(H) (CDCl₃) 7.37-7.27 (11H, m,Bn, 6-H), 5.87 (1H, s, 1′-H), 4.71 (2H, s, Bn), 4.64 (1H, d, J 12.0,Bn), 4.56 (1H, d, J 12.0, Bn), 4.36 (1H, t, J 5.7, 4′-H), 4.16 (1H, m,2″-H_(a)), 3.96 (1H, m, 2″-H_(b)), 3.74 (2H, m, 5′-H_(a), 5′-H_(b)),2.35-2.15 (2H, m, 1″-H_(a), 1″H_(b)), 1.88 (3H, s, CH₃). δ_(C) (CDCl₃)163.7 (C-4), 151.4 (C-2), 137.8, 137.3, 136.7 (Bn, C-6), 128.5, 128.4,128.0, 127.8, 127.5 (Bn), 109.9 (C-5), 108.6 (C-2′), 88.8, 87.1, 80.9(C-1′, C-3′, C-4′), 73.6, 68.5, 68.1, 67.9 (C-5′, C-2″, Bn), 30.9(C-1″), 12.6 (CH₃). FAB-MS: m/z 481.03 [M+H]⁺, 503.02 [M+Na]⁺ (Found: C,64.6; H, 5.8; N, 5.7; C₂₆H₂₈O₇N₂ requires C, 65.0; H, 5.9; N, 5.8%).

Example 16

(1S,5R,6R,8R)-1,5-Dihydroxy-6-hydroxymethyl-8-(thymin-1-yl)-2,7-dioxabicyclo-[3.3.0]octane(13). Compound 13 was successfully derived from compound 12 by catalyticremoval of the benzyl protecting group in the same way as described inpreparation of 6. Purification of 13 was accomplished by column silicagel chromatography eluting with gradient concentrations (6 to 14%) ofmethanol in dichloromethane. Analytical amounts of compound 13 (up to 15mg) were additionally purified by reverse-phase HPLC at column (10×250mm) packed by Nucleosil C18 (10 wn). Flow rate: 8 cm³/min; eluent: 0-10%acetonitrile in 60 min. Yield 82%. δ_(H) (CD₃OD) 7.44 (1H d, J 1.2,6-H), 5.83 (1H, s, 1′-H), 4.10-3.80 (5H, m, 5′-H_(a), 5′-H_(b),2″-H_(a), 2″-H_(b), 4′-H), 2.39-2.25 (1H, m, 1″-H_(a)), 2.00-1.90 (1H,m, 1″-H_(b)), 1.87 (3H, d, J 1.2, CH₃). δ_(C) (CD₃OD) 166.3 (C-4), 152.7(C-2), 139.8 (C-6), 110.0, 109.6 (C-2′, C-5), 87.8, 85.8, 84.6 (C-1′,C-3′, C-4′), 68.8, 61.6 (C-5′, C-2″), 35.6 (C-1″), 12.4 (CH₃). FAB-MS:m/z 301.03 [M+H]⁺ (Found: C, 46.6; H, 5.7; N, 8.5; C₁₂H₁₆O₇N₂ requiresC, 48.0; H, 5.4; N, 9.3%).

Example 17

(1S,5R,6R,8R)-5-Benzyloxy-6-benzyloxymethyl-1-methoxy-8-(3-N-methylthymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane(14),(1S,5R,6R,8R)-5-Benzyloxy-6-benzyloxymethyl-1-hydroxy-8-(3-N-methylthymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane(15) and(1S,5R,6R,8R)-5-Benzyloxy-6-benzyloxymethyl-1-methoxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane(16). A mixture of compound 12 (1.04 g, 2.16 mmol) and sodium hydride(171 mg of a 60% suspention in mineral oil, 4.30 mmol) was dissolved inanhydrous dichloromethane (4 cm³) during 10 min under stiffing. Methyliodide (1 cm³, 16 mmol) was added and the reaction mixture was incubatedat 36° C. for 23 h. After evaporation, the residue was purified bysilica gel column chromatography eluting with a gradient of 0.4-2.4%methanol in dichloromethane (v/v) to give products 14, 15 and 16 andstarting material 12 (212 mg, 20.5%). Compound 14 (47 mg, 4.3%). δ_(H)(CDCl₃) 7.25-7.37 (11H, m, Bn, 6-H), 6.15 (1H, s, 1′-H), 4.74 (1H, d, J11.5, Bn), 4.67 (1H, d, J 11.3, Bn), 4.62 (1H, d, J 12.1, Bn), 4.55 (1H,d, J 11.9, Bn), 4.34 (1H, t, J 5.6, 4′-H), 3.99, (1H, m, 2″-H_(a)), 4.22(1H, m, 2″-H_(b)), 3.72 (2H, m, 5′-H_(a), 5′-H_(a)), 3.41 (3H, s,CH₃—O), 3.35 (3H, s, CH₃—N³), 2.27 (1H, m, 1″-H_(a)), 2.41 (1H, m,1″-H_(b)), 1.93 (3H, s, 5-CH₃). δ_(C) (CDCl₃) 163.3 (C-4), 151.0 (C-2),138.2, 137.3, 135.7 (Bn, C-6), 128.3, 128.2, 127.8, 127.6, 127.4, 126.9(Bn), 111.8 (CS), 108.5 (C-2′), 89.1, 84.8, 79.5 (C-1′, C-3′, C-4′),73.5, 68.4, 68.2, 67.3 (Bn, C-5′, C-2″), 50.8 (CH₃—O), 32.6 (C-1″), 27.9(CH₃—N), 13.2 (CH₃). FAB-MS: m/z 508.88 [M+H]⁺ (Found: C, 65.7; H, 6.9;N, 4.8; C₂₈H₃₂O₇N₂ requires C, 66.1; H, 6.3; N, 5.5%). Compound 15 (97mg, 9.1%). δ_(H) (CDCl₃) 7.37-7.28 (11H, m, Bn, 6-H), 5.86 (1H, s,1′-H), 4.72 (2H, s, Bn), 4.64 (1H, d, J 11.9, Bn), 4.58 (1H, d, J 11.9,Bn), 4.37 (1H, t, J 5.6, 4′-H), 4.13 (1H, m, 2″-H_(a)), 3.93 (1H, m,2″-H_(b)), 3.75 (2H, m, 5′-H_(a), 5′-H_(b)), 3.34 (1H, s, CH₃—N),2.32-2.16 (2H, m, 1″-H_(a), 1″-H_(b)), 1.93 (3H, s, CH₃). δ_(C) (CDCl₃)163.2 (C-4), 151.9 (C-2), 137.5, 137.1, 134.0 (Bn, C-6), 128.4, 128.3,128.1, 127.9 127.7, 127.6, 127.3 (Bn), 108.8, 108.5 (C-2′, C-5), 88.7(C1′), 88.0, 81.0 (C-3′, C-4′), 73.5, 68.3, 67.9, 67.7 (Bn, C-5′, C-2″),30.6 (C-1″), 27.8 (CH₃—N), 13.2 (CH₃). FAB-MS m/z 495.28 [M+H]⁺, 517.24[M+Na]⁺. Compound 16 (665 mg, 62.3%). δ_(H) (CDCl₃) 7.35-7.25 (11H, m,Bn, 6-H), 6.06 (1H, s, 1′-H), 4.73 (1H, d, J 11.5, Bn), 4.66 (1H, d, J11.3, Bn), 4.61 (1H, d, J 11.9, Bn), 4.55 (1H, d, J 12.0, Bn), 4.34 (1H,t, J 5.6, 4′-H), 4.20 (1H, m, 2″-H_(a)), 3.98 (1H, m, 2″-H_(b)), 3.72(2H, m, 5′-H_(a), 5′-H_(b)), 3.40 (3H, s, CH₃—O), 2.45-2.35 (1H, m,1″-H_(a)), 2.30-2.20 (1H, m, 1″-H_(b)), 1.90 (3H, d, J 1.1, CH₃). δ_(C)(CDCl₃) 163.2 (C-4), 150.1 (C-2), 138.2, 137.9, 137.3 (Bn, C-6), 128.4,128.2, 127.8, 127.6 127.4, 127.1 (Bn), 110.8 (C-5), 109.3 (C-2′), 89.2,84.2, 79.6 (C-1′, C-3′, C-4′), 73.6, 68.5, 68.3, 67.4 (Bn, C-5′, C-2″),50.8 (CH₃—O), 32.6 (C-1″), 12.5 (CH₃). FAB-MS m/z 495.22 [M+H]⁺, 517.23[M+Na]⁺ (Found: C, 66.2; H, 7.2; N, 4.4; C₂₇H₃₀O₇N₂ requires C, 65.6; H,6.1; N, 5.7%).

Example 18

(1S,5R,6R,8R)-5-Hydroxy-6-hydroxymethyl-1-methoxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane(17). To a solution of nucleoside 16 (1.20 g, 2.43 mmol) in methanol (10cm³) was added 20% palladium hydroxide over charcoal (250 mg) and themixture was carefully degassed under reduced pressure. An atmosphere ofhydrogen was applied and stirring was continued for 12 h. The catalystwas removed by filtration of the reaction mixture through a glass column(1×8 cm) packed with silica gel in methanol. The column was additionallywashed with methanol (20 cm³). All fractions were collected, evaporatedto dryness and co-evaporated with petroleum ether to yield a glass-likesolid. This residue was purified by silica gel chromatography elutingwith a gradient of 5-10% methanol in dichloromethane (v/v). Thefractions containing the product were collected, combined and evaporatedto dryness. The residue was dissolved in anhydrous methanol (5 cm³) andanhydrous benzene (100 cm³) was added. Lyophilisation yielded nucleoside17 (0.61 g, 79%) as a white solid material. δ_(H) (CD₃OD) 7.45 (1H, s,6-H), 5.93 (1H, s, 1′-H), 4.15-3.81 (5H, m, 5′-H_(a), 5′-H_(b),2″-H_(a), 2″-H_(b), 4′-H), 3.43 (3H, s, CH₃—O), 2.47-2.40 (1H, m,1″-H_(a)), 2.03-1.93 (1H, m, 1″-H_(b)), 1.92 (3H, s, CH₃). δ_(C) (CD₃OD)164.1 (C-4), 150.1 (C-2), 138.3 (C-6), 109.6 (C-5), 108.3 (C-2′), 84.4,84.1, 82.4 (C-1′, C-3′, C-4′), 68.0, 59.5 (C-5′, C-2″), 49.6 (CH₃—O),34.0 (C-1″), 10.5 (CH₃). FAB-MS m/z 315.13 [M+H]⁺, 337.09 [M+Na]⁺(Found: C, 49.9; H, 5.7; N, 8.2; C₁₃H₁₈O₇N₂ requires C, 49.7; H, 5.8; N,8.9%).

Example 19

(1S,5R,6R,8R)-6-(4,4′-Dimethoxytrityloxymethyl)-5-hydroxy-1-methoxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane(18). A mixture of compound 17 (0.95 g, 3.03 mmol) and4,4′-dimethoxytrityl chloride (1.54 g, 4.77 mmol) was dissolved inanhydrous pyridine (20 cm³) and stirred for 4 h at room temperature. Thereaction mixture was evaporated to give an oily residue which wasco-evaporated with toluene (2×20 cm³). Dichloromethane (50 cm³) and asaturated aqueous solution of sodium hydrogencarbonate (50 cm³) wereadded, the organic phase was separated and evaporated, and the residuepurified by silica gel HPLC (the residue was dissolved in the minimumamount of dichloromethane containing 0.5% triethylamine (v/v) andapplied to the column equilibrated by the same solvent. The column waswashed (ethylacetate:petroleum ether:triethylamine; 15:84.5:0.5 (v/v/v,1000 cm³) and the product was eluted in a gradient of methanol (0-2%) indichloromethane containing 0.5% of triethylamine (v/v/v) to givecompound 18 (1.71 g, 92.8%) as white solid material. δ_(H) (CDCl₃)7.51-7.17 (10H, m, DMT, 6-H), 6.79-6.85 (4H, m, DMT), 6.04 (1H, s,1′-H), 4.12-3.98 (3H, m, 5′-H_(a), 5′-H_(b), 4′-H), 3.77 (6H, s,CH₃-DMT), 3.49 (3H, s, CH₃—O), 3.45-3.32 (2H, m, 2″-H_(a), 2″-H_(b)),2.11-2.01 (1H, m,1″-H_(a)), 1.94-1.87 (1H, m, 1″-H_(b)), 1.93 (3H, s,CH₃). δ_(C) (CDCl₃) 164.2 (C-4), 158.6, 144.7, 135.7, 130.1, 128.2,127.9, 126.8, 113.2 (DMT), 150.7 (C-2), 137.7 (C-6), 109.8, 109.7 (C-5,C-2′), 86.5, 85.3, 85.0, 81.4 (DMT, C-1′, C-3′, C-4′), 69.2, 62.4 (C-5′,C-2″), 55.2 (CH₃-DMT), 51.7 (CH₃—O), 35.5 (C-1″), 12.7 (CH₃). FAB-MS m/z617.26 [M+H]⁺, 639.23 [M+Na]⁺ (Found: C, 66.4; H, 6.1; N, 4.2;C₃₄H₃₆O₉N₂ requires C, 66.2; H, 5.9; N, 4.5%).

Example 20

(1S,5R,6R,8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-6-(4,4′-dimethoxytrityloxymethyl)-1-methoxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane(19). Compound 18 (1.2 g, 1.95 mmol) was dissolved in anhydrousdichloromethane (10 cm³). N,N-Diisopropylethylamine (1.35 cm³, 7.8 mmol)and 2-cyanoethyl-N,N-diisopropylphosphoramidochloridite (0.92 g, 3.9mmol) were added under stirring at room temperature. After 72 h, themixture was diluted to 100 cm³ by dichloromethane and washed by asaturated aqueous solution of sodium hydrogencarbonate (50 cm³). Theorganic phase was evaporated and applied to silica gel HPLC purificationusing a gradient of eluent B (petroleum ether:dichloromethane:ethylacetate:pyridine; 45:45:10:0.5; v/v/v) in eluent A (petroleumether:dichloromethane:pyridine; 50:50:0.5; v/v/v). The fractionscontaining the product were concentrated, co-evaporated with toluene (10cm³) and dried under reduced pressure. The residue was dissolved inanhydrous benzene (20 cm³) and precipitated by addition of this solutioninto anhydrous petroleum ether (400 cm³) under stirring. The resultingwhite solid was isolated by filtration and dried to give compound 19(0.96 g, 60.3%). δ_(P) (CDCl₃) 142.64, 142.52. FAB-MS m/z 817.26 [M+H]⁺,839.24 [M+Na]⁺ (Found: C, 62.8; H, 6.4; N, 6.9; C₄₃H₅₃O₁₀N₄P requires C,63.2; H, 6.5; N, 6.9%).

Example 21

1,2-O-Isopropylidene-3-C-vinyl-α-D-ribofuranose (20). A solution of5-O-t-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythro-pent-3-ulofuranose(Y. Yoshimura, T. Sano, A. Matsuda, T. Ueda, Chem. Pharm. Bull., 1988,36, 162) (6.05 g, 0.020 mol) in anhydrous THF (250 cm³) was stirred at0° C. and a 1 M solution of vinylmagnesium bromide in ether (44 cm³, 44mmol) was added dropwise. The reaction mixture was stirred at roomtemperature for 2 h, whereupon saturated aqueous ammonium chloride (200cm³) was added, and extraction was performed using dichloromethane(3×300 cm³). The combined extract was washed with brine (3×250 cm³) anddried (Na₂SO₄). The solvent was removed and the residue was redissolvedin anhydrous THF (225 cm³). To this mixture was added a 1 M solution oftetrabutylammonium fluoride in THF (22 cm³, 22 mmol), stiffing at roomtemperature was continued for 20 min whereupon the mixture wasevaporated under reduced pressure. The residue was dissolved indichloromethane (500 cm³) and washed with a saturated solution of sodiumhydrogencarbonate (2×200 cm³). The aqueous phase was extracted usingcontinuous extraction for 12 h and the combined extract was dried(Na₂SO₄) and evaporated. The residue was purified by silica gel columnchromatography using dichloromethane/methanol (99:1, v/v) as eluent togive furanose 20 as a white solid material (3.24 g, 75%). δ_(H) (CDCl₃)5.84 (1H, d, J 3.7, 1-H), 5.74 (1H, dd, J 11.0, 17.2, 1′-H), 5.52 (1H,dd, J 1.6, 17.1, 2′-H_(a)), 5.29 (1H, dd, J 1.3, 11.0, 2′-H_(b)), 4.21(1H, d, J 3.7, 2-H), 3.98 (1H, t, J 5.7, 4-H), 3.68-3.64 (2H, m,5-H_(a), 5-H_(b)), 2.88 (1H, s, 3-OH), 1.99 (1H, t, J 6.3, 5-OH), 1.60(3H, s, CH₃), 1.35 (3H, s, CH₃). δ_(C) (CDCl₃) 133.6 (C-1′), 116.2(C-2′), 113.0 (C(CH₃)₂), 103.8 (C-1), 83.4, 82.4 (C-4, C-2), 79.6 (C-3),61.3 (C-5), 26.5, 26.4 (CH₃).

Example 22

3,5-Di-O-benzyl-1,2-O-isopropylidene-3-C-vinyl-α-D-ribofuranose (21). A60% suspension of sodium hydride (w/w, 1.78 g, 44.5 mmol) in anhydrousDMF (50 cm³) was stirred at 0° C. and a solution of furanose 20 (3.20 g,14.8 mmol) in anhydrous DMF (35 cm³) was added dropwise over 30 min. Themixture was stirred at 50° C. for 1 h and subsequently cooled to 0° C. Asolution of benzyl bromide (5.3 mL, 44.5 mmol) in anhydrous DMF (5.3cm³) was added dropwise, and the mixture was stirred at room temperaturefor 20 h. The reaction mixture was evaporated and redissolved indichloromethane (300 cm³), washed with saturated aqueous sodiumhydrogencarbonate (3×200 cm³) and dried (Na₂SO₄). The solvents wereremoved under reduced pressure and the residue was purified by silicagel column chromatography using petroleum ether/ethylacetate (9:1, v/v)as eluent to give furanose 21 as a white solid material (5.36 g, 91%).δ_(H) (CDCl₃) 7.40-7.26 (10H, m, Bn), 5.90 (1H, d, J 3.6, 1-H), 5.72(1H, dd, J 11.1, 17.9, 1′-H), 5.41 (1H, dd, J 0.7, 11.1, 2′-H_(a)), 5.30(1H, dd, J 0.5, 17.8, 2′-H_(b)), 4.70-4.45 (6H, m, Bn, 2-H, 4-H), 3.69(1H, dd, J 2.6, 10.8, 5-H_(a)), 3.50 (1H, dd, J 7.9, 10.9, 5-H_(b)),1.64 (3H, s, CH₃), 1.40 (3H, s, CH₃). δ_(C) (CDCl₃) 138.6, 138.3 (Bn),134.5 (C-1′), 128.3-127.4 (Bn), 118.2 (C-2′), 112.9 (C(CH₃)₂), 104.7(C-1), 84.7, 81.1, 81.0 (C-2, C-3, C-4), 73.3 (C-5), 69.4, 67.0 (Bn),26.8, 26.6 (CH₃).

Example 23

1,2-Di-O-acetyl-3,5-di-O-benzyl-3-C-vinyl-α,β-D-ribofuranose (22). Asolution of furanose 21 (4.40 g, 11.1 mmol) in 80% aqueous acetic acid(50 cm³) was stirred at 90° C. for 8 h. The solvents were removed andthe residue was coevaporated with 99% ethanol (3×25 cm³), toluene (3×25cm³) and anhydrous pyridine (2×25 cm³) and redissolved in anhydrouspyridine (20 cm³). Acetic anhydride (17 cm³) was added and the solutionwas stirred at room temperature for 48 h. The reaction was quenched withice-cold water (100 cm³) and extracted with dichloromethane (2×100 cm³).The combined extract was washed with saturated aqueous sodiumhydrogencarbonate (3×100 cm³) and dried (Na₂SO₄). The solvent wasevaporated and the residue was purified by silica gel columnchromatography using petroleum ether/ethylacetate (4:1, v/v) as eluentto give furanose 22 as an oil (4.27 g, 87%, α:β˜1:1). δ_(C) (CDCl₃)169.9, 169.8 (C═O), 139.0, 138.6, 138.0, 137.8 (Bn), 133.3, 132.4(C-1′), 128.4-126.8 (Bn), 119.6, 119.5 (C-2′), 99.5, 94.0 (C-1), 85.4,85.0, 84.3, 83.6, 77.7, 73.6, 73.5, 73.3, 70.0, 69.2, 67.5, 67.2 (C-2,C-3, C-4, C-5, Bn), 21.0, 20.9, 20.6, 20.4 (CH₃).

Example 24

1-(2-O-Acetyl-3,5-di-O-benzyl-3-C-vinyl-β-D-ribofuranosyl)thymine (23).To a stirred solution of compound 22 (4.24 g, 9.6 mmol) and thymine(2.43 g, 19.3 mmol) in anhydrous acetonitrile (100 cm³) was addedN,O-bis(trimethylsilyl)acetamide (11.9 cm³, 48.1 mmol). The reactionmixture was stirred at reflux for 30 min. After cooling to 0° C.,trimethylsilyl triflate (3.2 cm³, 16.4 mmol) was added dropwise and thesolution was stirred for 24 h at room temperature. The reaction wasquenched with cold saturated aqueous sodium hydrogencarbonate (100 cm³)and the resulting mixture was extracted with dichloromethane (3×50 cm³).The combined extract was washed with saturated aqueous sodiumhydrogencarbonate (2×50 cm³) and brine (2×50 cm³) and dried (Na₂SO₄).The extract was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give nucleoside 23 asa white foam (4.03 g, 83%). δ_(H) (CDCl₃) 8.78 (1H, br s, NH), 7.75 (1H,s, 6-H), 7.38-7.26 (10H, m, Bn), 6.49 (1H, d, J 8.1, 1′-H), 5.99-5.88(2H, m, 2′-H and 1″-H), 5.54-5.48 (2H, m, 2″-H_(a), 2″-H_(b)), 4.91-4.50(4H, m, Bn), 4.34 (1H, s, 4′-H), 3.80 (1H, m, 5′-H_(a)), 3.54 (1H, m,5′-H_(b)), 2.11 (3H, s, COCH₃), 1.48 (3H, s, CH₃). δ_(C) (CDCl₃) 170.1(C═O), 163.8 (C-4), 151.0 (C-2), 138.9, 136.9 (Bn), 136.1 (C-6), 132.0(C-1″), 128.7, 128.5, 128.2, 127.8, 127.7, 127.5, 127.5, 127.1 (Bn),120.7 (C-2″), 111.3 (C-5), 85.4 (C-1′), 85.2 (C-3′), 84.3 (C-4′), 76.0(C-2′), 73.7 (C-5′), 69.3, 67.6 (Bn), 20.6 (COCH₃), 11.7 (CH₃). Found:C, 66.3; H, 6.0; N, 5.1; C₂₈H₃₀N₂O₇ requires C, 66.4; H, 6.0; N, 5.5%.

Example 25

1-(3,5-Di-O-benzyl-3-C-vinyl-β-D-ribofuranosyl)thymine (24). To astirred solution of nucleoside 23 (3.90 g, 7.7 mmol) in anhydrousmethanol (40 cm³) was added sodium methoxide (0.83 g, 15.4 mmol). Themixture was stirred at room temperature for 42 h and then neutralisedwith dilute aqueous hydrochloric acid. The mixture was extracted withdichloromethane (2×150 cm³), and the combined extract was washed withsaturated aqueous sodium hydrogencarbonate (3×100 cm³) and dried(Na₂SO₄). The solvent was removed under reduced pressure to givenucleoside 24 as a white foam (3.48 g, 97%). δ_(H) (CDCl₃) 8.89 (1H, brs, NH), 7.60 (1H, d, J 0.9, 6-H), 7.36-7.26 (10H, m, Bn), 6.23 (1H, d, J7.8, 1′-H), 5.98 (1H, dd, J 11.2, 17.7, 1″-H), 5.66 (1H, d, J 17.7,2″-H_(a)), 5.55 (1H, d, J 11.5, 2″-H_(b)), 4.75-4.37 (6H, m, 2′-H, 4′-H,Bn), 3.84 (1H, dd, J 2.7, 10.8, 5′-H_(a)), 3.58 (1H, d, J 11.2,5′-H_(b)), 3.23 (1H, d, J 10.6, 2′OH), 1.50 (3H, s, CH₃). δ_(C) (CDCl₃)163.7 (C-4), 151.3 (C-2), 138.0, 136.9 (Bn), 136.0 (C-6), 131.2 (C-1″),128.8, 128.6, 128.3, 127.8, 127.7, 127.3 (Bn), 120.7 (C-2″), 111.3(C-5), 87.3 (C-1′), 84.6 (C-3′), 81.4 (C-4′), 78.0 (C-2′), 73.7 (C-5′),70.0, 66.4 (Bn), 11.8 (CH₃). Found: C, 66.8; H, 6.2; N, 5.9; C₂₆H₂₈N₂O₆requires C, 67.2; H, 6.1; N, 6.0%.

Example 26

1-(3,5-Di-O-benzyl-2-O-methanesulfonyl-3-C-vinyl-β-D-ribofuranosyl)thymine(25). Nucleoside 24 (2.57 g, 5.53 mmol) was dissolved in anhydrouspyridine (18 cm³) and cooled to 0° C. Methanesulfonyl chloride (1.28cm³, 16.6 mmol) was added dropwise and the mixture was stirred at roomtemperature for 30 min. The reaction was quenched with water (5 cm³) andthe resulting mixture was extracted with dichloromethane (3×80 cm³). Thecombined extract was washed with saturated aqueous sodiumhydrogencarbonate (3×120 cm³) and dried (Na₂SO₄). The solvent wasevaporated under reduced pressure and the residue was purified by silicagel column chromatography using dichloromethane/methanol (99:1, v/v) aseluent to give nucleoside 25 as a yellow foam (2.53 g, 84%). δ_(H)(CDCl₃) 8.92 (1H, br s, NH), 7.71 (1H, d, J 1.4, 6-H), 7.41-7.28 (10H,m, Bn), 6.57 (1H, d, J 7.8, 1′-H), 5.99-5.61 (4H, m, 2′-H, 1″-H and2″-H_(a), 2″-H_(b)), 4.86-4.50 (4H, m, Bn), 4.37 (1H, dd, J 1.5, 2.4,4′-H), 8.82 (1H, dd, J 2.6, 11.0, 5′-H_(a)), 3.55 (1H, dd, J 1.2, 11.0,5′-H_(b)), 3.02 (3H, s, CH₃), 1.47 (3H, d, J 1.1, CH₃). δ_(C) (CDCl₃)163.7 (C-4), 151.5 (C-2), 138.7, 136.7 (Bn), 135.7 (C-6), 130.9 (C-1″),128.8, 128.5, 128.4, 127.6, 127.0 (Bn), 121.8 (C-2″), 111.9 (C-5), 85.1(C-1′), 84.5 (C-3′), 84.0 (C-4′), 80.7 (C-2′), 73.7 (C-5′), 69.2, 67.7(Bn), 38.9 (CH₃), 11.8 (CH₃).

Example 27

1-(3,5-Di-O-benzyl-3-C-vinyl-β-D-arabinofuranosyl)thymine (26). Asolution of nucleoside 25 (2.53 g, 4.66 mmol) in a mixture of ethanol(50 cm³), water (50 cm³) and 1 M aqueous sodium hydroxide (15 cm³) wasstirred under reflux for 16 h. The mixture was neutralised using diluteaqueous hydrochloric acid, the solvent was evaporated under reducedpressure, and the residue was extracted with dichloromethane (3×120cm³). The combined extract was washed with saturated aqueous sodiumhydrogencarbonate (3×150 cm³) and dried (Na₂SO₄). The solvent wasremoved under reduced pressure and the residue was purified by silicagel column chromatography using dichloromethane/methanol (99:1) aseluent to give 26 as a white foam (1.61 g, 74%). δ_(H) (CDCl₃) 9.89 (1H,br s, NH), 7.50 (1H, d, J1.1, 6-H), 7.41-7.26 (Bn), 6.28 (1H, d, J 2.8,1′-H), 6.05 (1H, dd, J 11.1, 17.9, 1″-H), 5.58-5.50 (2H, m, 2″-H_(a),2″-H_(b)), 4.98 (1H, d, J 9.0, 2′-OH), 4.64-4.31 (6H, m, 2′-H, 4′-H,Bn), 3.73 (2H, m, 5′-H_(a), 5′-H_(b)), 1.73 (1H, d, J 0.6, CH₃). δ_(C)(CDCl₃) 165.1 (C-4), 150.5 (C-2), 138.4, 138.0, 136.7 (C-6, Bn), 130.4(C-1″), 128.8, 128.6, 128.5, 128.1, 128.0, 127.8 (Bn), 120.6 (C-2″),108.1 (C-5), 88.6 (C-1′), 87.9 (C-3′), 87.2 (C-4′), 73.7 (C-2′), 71.8(C-5′), 69.7, 66.3 (Bn), 12.3 (CH₃). Found: C, 66.8; H, 6.2; N, 5.9;C₂₆H₂₈N₂O₆ requires C, 67.2; H, 6.1; N, 6.0.

Example 28

1-(3,5-Di-O-benzyl-3-C-hydroxymethyl-(3-D-arabinofuranosyl)thymine (27).To a solution of nucleoside 26 (2.00 g, 4.31 mmol) in a mixture of THF(15 cm³) and water (15 cm³) was added sodium periodate (2.76 g, 12.9mmol) and a 2.5% solution of osmium tetraoxide in t-butanol (w/w, 0.54cm³, 43 μmol). The reaction was stirred at room temperature for 18 h,quenched with water (50 cm³), and the mixture was extracted withdichloromethane (2×100 cm³). The combined extract was washed withsaturated aqueous sodium hydrogen carbonate (3×75 cm³), dried (Na₂SO₄)and evaporated under reduced pressure. The residue was redissolved in amixture of THF (15 cm³) and water (15 cm³), and sodium borohydride (488mg, 12.9 mmol) was added. The reaction mixture was stirred at roomtemperature for 1 h, water (50 cm³) was added, and the mixture wasextracted with dichloromethane (2×100 cm³). The combined organic phasewas washed with saturated aqueous sodium hydrogencarbonate (3×75 cm³)and dried (Na₂SO₄). The solvent was removed and the residue was purifiedby silica gel column chromatography using dichloromethane/methanol(98:2, v/v) as eluent to give nucleoside 27 as a white foam (732 mg,36%). δ_(H) (CDCl₃) 11.09 (1H, br s, NH), 7.41 (1H, d, J 1.0, 6-H),7.38-7.26 (Bn), 6.16 (1H, d, J 2.6, 1′-H), 5.12 (1H, d, J 5.4, 2′-OH),4.66-4.29 (6H, m, 2′-H, 4′-H, Bn), 4.02-3.96 (2H, m, 1″-H_(a),1″-H_(b))_(,) 3.90 (1H, dd, J 7.2, 9.7, 5′-H_(a)), 3.79 (1H, dd, J 5.6,9.7, 5′-H_(b)), 2.49 (1H, t, J 6.4, 1″-OH), 1.68 (3H, d, J 0.6, CH₃);δ_(C) (CDCl₃) 166.1 (C-4), 150.6 (C-2), 139.0, 137.9, 137.0 (C-6, Bn),128.7, 128.6, 128.4, 128.3, 128.0 (Bn), 107.5 (C-5), 88.2 (C-1′), 88.1(C-3′), 84.2 (C-4′), 73.7 (C-5′), 72.1 (C-2′), 69.3, 65.4 (Bn), 58.6(C-1″), 12.3 (CH₃).

Example 29

(1R,2R,4R,5S)-1-Benzyloxy-2-benzyloxymethyl-4-(thymin-1-yl)-3,6-dioxabicyclo-[3.2.0]heptane(28). A solution of compound 27 (2.26 g, 4.83 mmol) in anhydrouspyridine (20 cm³) was stirred at −40° C. and a solution ofmethanesulphonyl chloride (0.482 cm³, 4.83 mmol) in anhydrous pyridine(10 cm³) was added. The reaction mixture was stirred at room temperaturefor 17 h, water (50 cm³) was added, and the mixture was extracted withdichloromethane (2×100 cm³). The combined organic phase was washed withsaturated aqueous sodium hydrogencarbonate (3×100 cm³), dried (Na₂SO₄)and evaporated under reduced pressure. The residue was purified bysilica gel column chromatography using dichloromethane/methanol (99:1,v/v) as eluent to give an intermediate which after evaporation of thesolvents was dissolved in anhydrous DMF (15 cm³). This solution wasadded dropwise to a suspension of 60% sodium hydride (461 mg, 11.5 mmol)in anhydrous DMF (15 cm³) at 0° C. The reaction was stirred at roomtemperature for 30 min, then quenched with water (60 cm³). Afterneutralisation using dilute aqueous hydrochloric acid, the mixture wasdissolved in dichloromethane (150 cm³), washed with saturated aqueoussodium hydrogencarbonate (3×100 cm³) and dried (Na₂SO₄). The solventswere evaporated and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (99:1, v/v) as eluent togive nucleoside 28 as a white foam (2.00 g, 93%). δ_(H) (CDCl₃) 9.13(1H, br s, NH), 7.55 (1H, d, J 1.4, 6-H), 7.40-7.26 (Bn), 5.99 (1H, d, J2.5, 1′-H), 5.30 (1H, d, J 2.7, 2′-H), 4.88-4.57 (6H, m, 1″-H_(a),1″-H_(b), Bn), 4.22-4.19 (1H, m, 4′-H), 3.92 (1H, dd, J 6.2, 10.8,5′-H_(a)), 3.82 (1H, dd, J 3.7, 10.8, 5′-H_(b)), 1.91 (3H, d, J 1.3,CH₃). δ_(C) (CDCl₃) 163.8 (C-4), 150.3 (C-2), 137.6 (C-6), 137.5, 137.0(Bn), 128.7, 128.6, 128.2, 128.0, 127.8, 127.3 (Bn), 109.8 (C-5), 85.7(C-3′), 84.1 (C-1′), 83.5 (C-4′), 79.7 (C-1″), 73.9 (C-2′), 73.6 (C-5′),68.6, 67.8 (Bn), 12.4 (CH₃). FAB m/z 451 [M+H]⁺, 473 [M+Na]⁺. Found: C,66.3; H, 5.9; N, 6.1; C₂₅H₂₆N₂O₆ requires C, 66.7; H, 5.8; N, 6.2%.

Example 30

(1R,2R,4R,5S)-1-Hydroxy-2-hydroxymethyl-4-(thymin-1-yl)-3,6-dioxabicyclo-[3.2.0]heptane(29). To a stirred solution of nucleoside 28 (180 mg, 0.40 mmol) inethanol (3 cm³) was added 10% palladium hydroxide over carbon (90 mg).The mixture was degassed several times with argon and placed under ahydrogen atmosphere. The reaction mixture was stirred at roomtemperature for 6 h, then filtered through celite. The filtrate wasevaporated under reduced pressure and the residue was purified by silicagel column chromatography using dichloromethane/methanol (96:4, v/v) aseluent to give nucleoside 29 as a white solid material (92 mg, 86%).δ_(H) (CD₃OD) 7.79 (1H, d, J 1.2, 6-H), 5.91 (1H, d, J 2.5, 1′-H), 4.96(1H, d, J 2.5, 2′-H), 4.92 (1H, d, J7.4, 1″-H_(a)), 4.58 (1H, dd, J 0.9,7.4, 1″-H_(b)), 3.98 (1H, dd, J 7.3, 12.8, 5′-H_(a)), 3.87-3.82 (2H, m,4′-H, 5′-H_(b)), 3.34 (2H, s, 3′-OH, 5′-OH), 1.87 (3H, d, J 1.3, CH₃).δ_(C) (CD₃OD) 166.5 (C-4), 152.1 (C-2), 140.1 (C-6), 110.1 (C-5), 91.2(C-2′), 85.1 (C-1′), 84.0 (C-4′), 79.6 (C-3′), 78.6 (C-1″), 61.1 (C-5′),12.3 (CH₃).

Example 31

(1R,2R,4R,5S)-1-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-2-(4,4′-dimethoxytrityloxymethyl)-4-(thymin-1-yl)-3,6-dioxabicyclo[3.2.0]heptane(30). To a solution of diol 29 (250 mg, 0.925 mmol) in anhydrouspyridine (4 cm³) was added 4,4′-dimethoxytrityl chloride (376 mg, 1.11mmol) and the mixture was stirred at room temperature for 18 h. Thereaction was quenched with methanol (1.5 cm³) and the mixture wasevaporated under reduced pressure. A solution of the residue indichloromethane (30 cm³) was washed with saturated aqueous sodiumhydrogencarbonate (3×20 cm³), dried (Na₂SO₄) and evaporated. The residuewas purified by silica gel column chromatography usingdichloromethane/methanol (98:2, v/v) as eluent to give an intermediatewhich was dissolved in anhydrous dichloromethane (7.0 cm³).N,N-Diisopropylethylamine (0.64 cm³, 3.70 mmol) followed by 2-cyanoethylN,N-diisopropylphosphoramidochloridite (0.41 cm³, 1.85 mmol) were addedand the mixture was stirred at room temperature for 25 h. The reactionwas quenched with methanol (3 cm³), and the mixture was dissolved inethylacetate (70 cm³), washed with saturated aqueous sodiumhydrogencarbonate (3×50 cm³) and brine (3×50 cm³), dried (Na₂SO₄), andwas evaporated under reduced pressure. The residue was purified bysilica gel column chromatography using petroleumether/dichloromethane/ethylacetate/triethylamine (100:45:45:10, v/v/v/v)as eluent. The residue obtained was dissolved in toluene (2 cm³) andprecipitated under stiffing from petroleum ether at −50° C. Afterevaporation of the solvents, the residue was coevaporated with anhydrousacetonitrile (4×5 cm³) to give 30 as a white foam (436 mg, 61%). ³¹P NMR(CDCl₃) 146.6.

Example 32

3,5-Di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose(31). To a solution of3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (R.D. Youssefyeh, J. P. H. Verheyden and J. G. Moffatt, J. Org. Chem.,1979, 44, 1301) (20.1 g, 0.064 mol) in anhydrous DMF (100 cm³) at −5° C.was added a suspension of NaH (60% in mineral oil (w/w), four portionsduring 1 h 30 min, total 2.85 g, 0.075 mol). Benzyl bromide (8.9 cm³,0.075 mol) was added dropwise and stirring at room temperature wascontinued for 3 h whereupon ice-cold water (50 cm³) was added. Themixture was extracted with EtOAc (4×100 cm³) and the combined organicphase was dried (Na₂SO₄). After evaporation, the residue was purified bysilica gel column chromatography eluting with 5% EtOAc in petroleumether (v/v) to yield compound 31 (18.5 g, 71%). δ_(C) (CDCl₃) 138.0,137.4, 128.5, 128.3, 128.0, 127.8, 127.6 (Bn), 113.5 (C(CH₃)₂), 104.4(C-1), 86.5 (C-4), 78.8, 78.6 (Bn), 73.6, 72.6, 71.6 (C-2, C-3, C-5),63.2, (C-1′), 26.7, 26.1 (CH₃).

Example 33

4-C-(Acetoxymethyl)-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose(32). To a solution of furanose 31 (913 mg, 2.28 mmol) in anhydrouspyridine (4.5 cm³) was dropwise added acetic anhydride (1.08 cm³, 11.4mmol) and the reaction mixture was stirred at room temperature for 3 h.The reaction was quenched by addition of ice-cold water (50 cm³) andextraction was performed with dichloromethane (3×50 cm³). The combinedorganic phase was washed with a saturated aqueous solution of sodiumhydrogencarbonate (2×50 cm³), dried (Na₂SO₄) and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography using dichloromethane as eluent to give compound 32 as aclear oil (911 mg, 90%). δ_(H) (CDCl₃) 7.34-7.25 (10H, m, Bn), 5.77 (1H,d, J 3.6, 1-H), 4.78-4.27 (8H, m, Bn, H-5_(a), H-5_(b), H-3, H-2), 3.58(1H, d, J 10.3, H-1′_(a)), 3.48 (1H, d, J 10.5, H-1′_(b)), 2.04 (3H, s,COCH₃), 1.64 (3H, s, CH₃), 1.34 (3H, s, CH₃). (CDCl₃) 171.1 (C═O),138.2, 137.9, 128.6, 128.1, 128.0, 128.0, 127.8 (Bn), 114.0 (C(CH₃)₂),104.5 (C-1), 85.4 (C-4), 79.3, 78.6 (C-2, C-3), 73.7, 72.7, 71.2 (Bn,C-5), 64.9 (C-1′), 26.7, 26.3 (C(CH₃)₂), 21.0 (COCH₃). Found: C, 67.0;H, 6.5; C₂₅H₃₀O₇,¼H₂O requires C, 67.2; H, 6.9%.

Example 34

4-C-(Acetoxymethyl)-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribofuranose (33).A solution of furanose 32 (830 mg, 1.88 mmol) in 80% acetic acid (10cm³) was stirred at 90° C. for 4 h. The solvent was removed underreduced pressure and the residue was coevaporated with ethanol (3×5cm³), toluene (3×5 cm³) and anhydrous pyridine (3×5 cm³), and wasredissolved in anhydrous pyridine (3.7 cm³). Acetic anhydride (2.85 cm³)was added and the solution was stirred for 72 h at room temperature. Thesolution was poured into ice-cold water (20 cm³) and the mixture wasextracted with dichloromethane (2×20 cm³). The combined organic phasewas washed with a saturated aqueous solution of sodium hydrogencarbonate(2×20 cm³), dried (Na₂SO₄) and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography usingdichloromethane as eluent to give 33 (β:α˜1:3) as an clear oil (789 mg,86%). (CDCl₃) 171.0, 170.3, 170.0, 169.3 (C═O), 138.1, 137.6, 136.3,128.9, 128.6, 128.2, 128.0, 128.0, 127.9, 127.7, 124.0 (Bn), 97.8, 97.8(C1), 87.0, 85.0, 78.9, 74.5, 74.4, 73.8, 73.6, 72.0, 71.8, 71.0, 70.9,64.6, 64.4 (C-2, C-3, C-4, Bn, C-5, C-1′), 21.0, 20.8, 20.6 (COCH₃).Found: C, 64.2; H, 6.3; C₂₆H₃₀O₉ requires C, 64.2; H, 6.2%.

Example 35

1-(4-C-(Acetoxymethyl)-2-O-acetyl-3,5-di-O-benzyl-β-D-ribofuranosyl)thymine(34). To a stirred solution of the anomeric mixture 33 (736 mg, 1.51mmol) and thymine (381 mg, 3.03 mmol) in anhydrous acetonitrile (14.5cm³) was added N,O-bis(trimethylsilyl)acetamide(2.61 cm³, 10.6 mmol).The reaction mixture was stirred at reflux for 1 h, then cooled to 0° C.Trimethylsilyl triflate (0.47 cm³, 2.56 mmol) was added dropwise understirring and the solution was stirred at 65° C. for 2 h. The reactionwas quenched with a cold saturated aqueous solution of sodium hydrogencarbonate (15 cm³) and extraction was performed with dichloromethane(3×10 cm³). The combined organic phase was washed with saturated aqueoussolutions of sodium hydrogencarbonate (2×10 cm³) and brine (2×10 cm³),and was dried (Na₂SO₄). The solvent was removed under reduced pressureand the residue was purified by silica gel column chromatography usingdichloromethane/methanol (98:2, v/v) as eluent to give nucleoside 34 asa white solid material (639 mg, 76%). δ_(H) (CDCl₃) 8.98 (1H, br s, NH),7.39-7.26 (11H, m, Bn, 6-H), 6.22 (1H, d, J 5.3, 1′-H), 5.42 (1H, t, J5.4, 2′-H), 4.63-4.43 (5H, m, 3′-H, Bn), 4.41 (1H, d, J 12.2, 5′-H_(a)),4.17 (1H, d, J 12.1, 5′-H_(b)), 3.76 (1H, d, J 10.2, 1″-H_(a)), 3.51(1H, d, J 10.4, 1″-H_(b)), 2.09 (3H, s, COCH₃), 2.03 (3H, s, COCH₃),1.53 (3H, d, J 0.9, CH₃). (CDCl₃) 170.8, 170.4 (C═O), 163.9 (C-4), 150.6(C-2), 137.4 (C-6) 137.4, 136.1, 128.9, 128.8, 128.4, 128.2, 127.9 (Bn),111.7 (C-5), 87.2, 87.2, 86.1 (C-1′, C-3′, C-4′), 77.6 (C-2′), 74.8,73.9, 71.1, 63.8 (Bn, C-1″, C-5′), 20.9, 20.8 (COCH₃), 12.0 (CH₃).FAB-MS m/z 553 [M+H]⁺. Found: C, 62.7; H, 5.9; N, 4.7; C₂₉H₃₂N₂O₉requires C, 63.0; H, 5.8; N, 5.1%.

Example 36

1-(3,5-Di-O-benzyl-4-C-(hydroxymethyl)-β-D-ribofuranosyl)thymine (35).To a stirred solution of nucleoside 34 (553 mg, 1.05 mmol) in methanol(5.5 cm³) was added sodium methoxide (287 mg, 5.25 mmol). The reactionmixture was stirred at room temperature for 10 min, then neutralisedwith dilute hydrochloric acid. The solvent was partly evaporated andextraction was performed with dichloromethane (2×20 cm³). The combinedorganic phase was washed with saturated aqueous sodium hydrogencarbonate(3×20 cm³) and was dried (Na₂SO₄). The solvent was removed under reducedpressure to give 35 as a white solid material (476 mg, 97%). δ_(H)(CDCl₃) 7.47 (1H, d, J 1.0 6-H), 7.36-7.22 (10H, m, Bn), 6.07 (1H, d, J3.8, 1′-H), 4.87 (1H, d, J 11.7, Bn), 4.55 (1H, d, J 11.7, Bn),4.50-4.32 (4H, m, Bn, 2′-H, 3′-H), 3.84-3.53 (4H, m, 5′-H_(a), 5′-H_(b),1″-H_(a), 1″-H_(b)), 1.50 (3H, d, J 1.1, CH₃). (CDCl₃) 164.3 (C-4),151.3 (C-2), 137.6 (C-6) 136.4, 136.3, 128.8, 128.6, 128.4, 128.3, 127.9(Bn), 111.1 (C-5), 91.1, 91.0, 88.1 (C-1′, C-3′, C-4′), 77.4 (C-2′),74.8, 73.8, 71.4, 63.2 (Bn, C-5′, C-1″), 12.0 (CH₃). FAB-MS m/z 491[M+Na] ⁺. Found: C, 63.4; H, 6.0; N, 5.5; C₂₅H₂₈N₂O₇,¼H₂O requires C,63.5; H, 6.1; N, 5.9%.

Example 37

Intermediate 35A. A solution of nucleoside 35 (225 mg, 0.48 mmol) inanhydrous pyridine (1.3 cm³) was stirred at 0° C. and p-toluenesulphonylchloride (118 mg, 0.62 mmol) was added in small portions. The solutionwas stirred at room temperature for 16 h and additionalp-toluenesulphonyl chloride (36 mg, 0.19 mmol) was added. After stiffingfor another 4 h and addition of ice-cold water (15 cm³), extraction wasperformed with dichloromethane (2×15 cm³). The combined organic phasewas washed with saturated aqueous sodium hydrogencarbonate (3×15 cm³)and dried (Na₂SO₄). The solvent was removed under reduced pressure andthe residue was purified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give a intermediate35A (140 mg) which was used without further purification in the nextstep.

Example 38

(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-yl)-2,5-dioxabi-cyclo[2.2.1]heptane(36). Intermediate 35A (159 mg) was dissolved in anhydrous DMF (0.8cm³). The solution was added dropwise to a stirred suspension of 60%sodium hydride in mineral oil (w/w, 32 mg, 0.80 mmol) in anhydrous DMF(0.8 cm³) at 0° C. The mixture was stirred at room temperature for 72 hand then concentrated under reduced pressure. The residue was dissolvedin dichloromethane (10 cm³), washed with saturated aqueous sodiumhydrogencarbonate (3×5 cm³) and dried (Na₂SO₄). The solvent was removedunder reduced pressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (99:1, v/v) as eluent togive the bicyclic nucleoside 36 as a white solid material (65.7 mg,57%). δ_(H) (CDCl₃) 9.24 (1H, br s, NH), 7.49 (1H, s, 6-H), 7.37-7.26(10H, m, Bn), 5.65 (1H, s, 1′-H), 4.70-4.71 (5H, m, Bn, 2′-H), 4.02-3.79(5H, m, 3′-H, 5′-H_(a), 5′-H_(b), 1″-H_(a), 1″-H_(b)), 1.63 (3H, s,CH₃). (CDCl₃) 164.3 (C-4), 150.1 (C-2), 137.7, 137.1 (Bn), 135.0 (C-6),128.8, 128.7, 128.4, 128.0, 127.9 (Bn), 110.4 (C-5), 87.5, 87.3 (C-1′,C-3′), 76.7, 75.8, 73.9, 72.3, 72.1 (Bn, C-5′, C-2′, C-4′), 64.5 (C-1″),12.3 (CH₃). FAB-MS m/z 451 [M+H]⁺.

Example 39

(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo-[2.2.1]heptane(37). A solution of nucleoside 36 (97 mg, 0.215 mmol) in ethanol (1.5cm³) was stirred at room temperature and 20% palladium hydroxide overcarbon (50 mg) was added. The mixture was degassed several times withargon and placed in a hydrogen atmosphere with a baloon. After stiffingfor 4 h, the mixture was purified by silica gel column chromatographyusing dichloromethane-methanol (97:3, v/v) as eluent to give nucleoside37 as a white solid material (57 mg, 98%). δ_(H) ((CD₃)₂SO) 11.33 (1H,br s, NH), 7.62 (1H, d, J 1.1 Hz, 6-H), 5.65 (1H, d, J 4.4 Hz, 3′-OH),5.41 (1H, s, 1′-H), 5.19 (1H, t, J 5.6 Hz, 5′-OH), 4.11 (1H, s, 2′-H),3.91 (1H, d, J 4.2 Hz, 3′-H), 3.82 (1H, d, J 7.7 Hz, 1″-H_(a)), 3.73(1H, s, H′-5_(a)), 3.76 (1H, s, 5′-H_(b)), 3.63 (1H, d, J 7.7 Hz,1″-H_(b)), 1.78 (3H, d, J 0.7 Hz, CH₃). (CDCl₃) 166.7 (C-4), 152.1(C-2), 137.0 (C-6), 110.9 (C-5), 90.5, 88.4 (C-1′, C-4′), 80.9, 72.5,70.4 (C-2′, C-3′, C-5′), 57.7 (C-1″), 12.6 (CH₃). E1-MS m/z 270 [M]⁺.

Example 40

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(38). To a solution of nucleoside 37 (1.2 g, 4.44 mmol) in anhydrouspyridine (5 cm³) was added 4,4′-dimethoxytrityl chloride (2.37 g, 7.0mmol) at 0° C. The solution was stirred at room temperature for 2 hwhereupon the reaction was quenched with ice-cold water (10 cm³) andextracted with dichloromethane (3×15 cm³). The combined organic phasewas washed with saturated aqueous solutions of sodium hydrogen carbonate(3×10 cm³), brine (2×10 cm³) and dried (Na₂SO₄). The solvent was removedunder reduced pressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (98:2, v/v) as eluent togive nucleoside 38 as a white solid material (2.35 g, 93%). δ_(H)(CDCl₃) 9.89 (1H, br s, NH), 7.64 (1H, s, 6-H), 7.47-7.13 (9H, m, DMT),6.96-6.80 (4H, m, DMT), 5.56 (1H, s, 1′-H), 4.53 (1H, br s, 2′-H), 4.31(1H, m, 3′-H), 4.04-3.75 (9H, m, 1″-H_(a), 1″-H_(b), 3′-OH, OCH₃), 3.50(2H, br s, 5′-H_(a), 5′-H_(b)), 1.65 (3H, s, CH₃). δ_(C)(CDCl₃) 164.47(C-4), 158.66 (DMT), 150.13 (C-2), 144.56, 135.46, 135.35, 134.78,130.10, 129.14, 128.03, 127.79, 127.05 (C-6, DMT), 113.32, 113.14 (DMT),110.36 (C-5), 89.17, 88.16, 87.05 (C-1′, C-4′, DMT), 79.36, 71.81,70.25, 58.38 (C-2′, C-3′, C-5′, C-1″), 55.22 (OCH₃), 12.57 (CH₃). FAB-MSm/z 595 [M+Na]⁺, 573 [M+H]⁺.

Example 41

(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(39). To a solution of nucleoside 38 (2.21 g, 3.86 mmol) in anhydrousdichloromethane (6 cm³) at room temperature was addedN,N-diisopropylethylamine (4 cm³) and 2-cyanoethylN,N-diisopropylphosphoramidochloridite (1 cm³, 4.48 mmol) and stiffingwas continued for 1 h. MeOH (2 cm³) was added, and the mixture wasdiluted with ethyl acetate (10 cm³) and washed successively withsaturated aqueous solutions of sodium hydrogencarbonate (3×5 cm³) andbrine (3×5 cm³) and was dried (Na₂SO₄). The solvent was evaporated underreduced pressure, and the residue was purified by basic alumina columnchromatography with dichloromethane/methanol (99:1, v/v) as eluent togive 39 as a white foam. This residue was dissolved in dichloromethane(2 cm³) and the product was precipitated from petroleum ether (100 cm³,cooled to −30° C.) under vigorous stirring. The precipitate wascollected by filtration, and was dried to give nucleoside 39 as a whitesolid material (2.1 g, 70%). δ_(P)(CDCl₃) 149.06, 148.74. FAB-MS m/z 795[M+Na]⁺, 773 [M+H]⁺.

Example 42

1-(2-O-Acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-β-D-ribofuranosyl)uracil(40). To a stirred solution of the anomeric mixture 33 (3.0 g, 6.17mmol) and uracil (1.04 g, 9.26 mmol) in anhydrous acetonitrile (65 cm³)was added N,O-bis(trimethylsilyl)acetamide(9.16 cm³, 37.0 mmol). Thereaction mixture was stirred for 1 h at room temperature and cooled to0° C. Trimethylsilyl triflate (1.8 cm³, 10.0 mmol) was added dropwiseand the solution was stirred at 60° C. for 2 h. The reaction wasquenched by addition of a saturated aqueous solution of sodiumhydrogencarbonate (10 cm³) at 0° C. and extraction was performed withdichloromethane (3×20 cm³). The combined organic phase was washed withbrine (2×20 cm³) and was dried (Na₂SO₄). The solvents were removed underreduced pressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (99:1, v/v) as eluent togive nucleoside 40 as a white solid material (2.5 g, 75%). δ_(H) (CDCl₃)9.57 (1H, br s, NH), 7.63 (1H, d, J 8.2, 6-H), 7.40-7.24 (10H, m, Bn),6.18 (1H, d, J 4.5, 1′-H), 5.39-5.32 (2H, m, 2′-H, 5-H), 4.61 (1H, d, J11.6, Bn), 4.49-4.40 (5H, m, 3′-H, Bn, 1″-H_(a)), 4.37 (1H, d, J 12.3,1″-H_(b)), 3.76 (1H, d, J 10.1, 5′-H_(a)), 3.49 (1H, d, J 10.1,5′-H_(b)), 2.09 (s, 3H, COCH₃), 2.04 (3H, s, COCH₃). δ_(C) (CDCl₃)170.47, 169.94 (C═O), 163.32 (C-4), 150.30 (C-2), 140.24 (C-6), 137.15,136.95, 128.65, 128.52, 128.32, 128.19, 128.02, 127.77 (Bn), 102.57(C-5), 87.41, 86.14 (C-1′, C-4′), 77.09, 74.84, 74.51, 73.75, 70.60,63.73 (C-2′, C-3′, C-5′, C-1″, Bn), 20.79, 20.68 (COCH₃). FAB-MS m/z 539[M]⁺.

Example 43

1-(3,5-Di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl)uracil (41). To astirred solution of nucleoside 40 (2.0 g, 3.7 mmol) in methanol (25 cm³)was added sodium methoxide (0.864 g, 95%, 16.0 mmol). The reactionmixture was stirred at room temperature for 10 min and neutralised with20% aqueous hydrochloric acid. The solvent was partly evaporated and theresidue was extracted with ethyl acetate (3×50 cm³). The combinedorganic phase was washed with a saturated aqueous solution of sodiumhydrogencarbonate (3×20 cm³) and was dried (Na₂SO₄). The solvent wasremoved under reduced pressure and the residue was purified by silicagel column chromatography using dichloromethane/methanol (98.5:1.5, v/v)as eluent to give 41 as a white solid material (1.58 g, 95%). δ_(H)(CDCl₃) 9.95 (1H, br s, NH), 7.69 (d, J 8.1, 6-H), 7.35-7.17 (10H, m,Bn), 6.02 (1H, d, J 2.3, 1′-H), 5.26 (1H, d, J 8.1, 5-H), 4.80 (1H, d, J11.7, Bn), 4.47 (1H, d, J 11.7, Bn), 4.45-4.24 (4H, m, Bn, 2′-H, 3′-H),3.81 (1H, d, J 11.9, 1″-H_(a)), 3.69 (2H, br s, 2′-OH, 1″-OH), 3.67 (2H,m, 5′-H_(a), 1″-H_(b)), 3.48 (1H, d, J 10.3, 5′-H_(b)). δ_(C) (CDCl₃)163.78 (C-4), 150.94 (C-2), 140.61 (C-6), 137.33, 137.22, 128.59,128.18, 128.01 (Bn), 102.16 (C-5), 91.46, 88.36 (C-1′, C-4′), 76.73,74.66, 73.71, 73.29, 70.81, 62.81 (C-2′, C-3′, C-5′, C-1″, Bn). FAB-MSm/z 455 [M+H]⁺.

Example 44

Intermediate 42. A solution of nucleoside 41 (1.38 g, 3.0 mmol),anhydrous pyridine (2 cm³) and anhydrous dichloromethane (6 cm³) wasstirred at −10° C. and p-toluenesulfonyl chloride (0.648 g, 3.4 mmol)was added in small portions during 1 h. The solution was stirred at −10°C. for 3 h. The reaction was quenched by addition of ice-cold water (10cm³) and the mixture was extracted with dichloromethane (3×50 cm³). Thecombined organic phase was washed with a saturated aqueous solution ofsodium hydrogencarbonate (3×20 cm³) and was dried (Na₂SO₄). The solventwas removed under reduced pressure and the residue was purified bysilica gel column chromatography using dichloromethane/methanol (99:1,v/v) as eluent to give intermediate 42 (0.9 g) which was used withoutfurther purification in the next step.

Example 45

(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo-[2.2.1]heptane(43). Compound 42 (0.7 g) was dissolved in anhydrous DMF (3 cm³) and a60% suspension of sodium hydride (w/w, 0.096 g, 24 mmol) was added infour portions during 10 min at 0° C., and the reaction mixture wasstirred at room temperature for 12 h. The reaction was quenched withmethanol (10 cm³), and the solvents were removed under reduced pressure.The residue was dissolved in dichloromethane (20 cm³), washed withsaturated aqueous sodium hydrogencarbonate (3×6 cm³) and was dried(Na₂SO₄). The solvent was removed under reduced pressure and the residuewas purified by silica gel column chromatography usingdichloromethane/ethanol (99:1, v/v) as eluent to give nucleoside 43(0.30 g, 60%). δ_(H) (CDCl₃) 9.21 (1H, br s, NH), 7.70 (1H, d, J 8.2,6-H), 7.37-7.24 (10H, m, Bn), 5.65 (1H, s, 1′-H), 5.52 (1H, d, J 8.2,5-H), 4.68-4.45 (5H, m, 2′-H, Bn), 4.02-3.55 (5H, m, 3′-H, 5′-H_(a),1″-H_(a), 5′-H_(b), 1″-H_(b)). δ_(C) (CDCl₃) 163.33 (C-4), 149.73 (C-2),139.18 (C-6), 137.46, 136.81, 128.58, 128.54, 128.21, 128.10, 127.79,127.53 (Bn), 101.66 (C-5), 87.49, 87.33 (C-1′, C-4′), 76.53, 75.71,73.77, 72.33, 72.00, 64.35 (C-2′, C-3′, C-5′, C-1″, Bn). FAB-MS m/z 459[M+Na]⁺.

Example 46

(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo-[2.2.1]heptane(44). To a solution of compound 43 (0.35 g, 0.8 mmol) in absoluteethanol (2 cm³) was added 20% palladium hydroxide over carbon (0.37 g)and the mixture was degassed several times with hydrogen and stirredunder the atmosphere of hydrogen for 4 h. The solvent was removed underreduced pressure and the residue was purified by silica gel columnchromatography using dichloramethane/methanol (9:1, v/v) as eluent togive nucleoside 44 as a white solid material (0.16 g, 78%). δ_(H)(CD₃OD) 7.88 (1H, d, J 8.1, 6-H), 5.69 (1H, d, J 8.1, 5-H), 5.55 (1H, s,1′-H), 4.28 (1H, s, 2′-H), 4.04 (1H, s, 3′-H), 3.96 (1H, d, J 7.9,1″-H_(a)), 3.91 (2H, s, 5′-H), 3.76 (1H, d, J 7.9, 1″-H_(b)). δ_(C)(CD₃OD) 172.95 (C-4), 151.82 (C-2), 141.17 (C-6), 101.97 (C-5), 90.52,88.50 (C-1′, C-4′), 80.88, 72.51, 70.50, 57.77 (C-2′, C-3′, C-5′, C-1″).FAB-MS m/z 257 [M+H]⁺.

Example 47

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(45). To a solution of compound 44 (0.08 g, 0.31 mmol) in anhydrouspyridine (0.5 cm³) was added 4,4′-dimethoxytrityl chloride (0.203 g, 0.6mmol) at 0° C. and the mixture was stirred at room temperature for 2 h.The reaction was quenched with ice-cold water (10 cm³) and extractedwith dichloromethane (3×4 cm³). The combined organic phase was washedwith saturated aqueous solutions of sodium hydrogencarbonate (3×3 cm³)and brine (2×3 cm³) and was dried (Na₂SO₄). The solvent was removedunder reduced pressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (98:2, v/v) as eluent togive nucleoside 45 as a white solid material (0.12 g, 69%). δ_(H)(CDCl₃) 9.25 (1H, br s, NH), 7.93 (1H, d, J 7.2, 6-H), 7.50-7.15 (9H, m,DMT), 6.88-6.78 (4H, m, DMT), 5.63 (1H, s, 1′-H), 5.59 (1H, d, J 8.0,5-H), 4.48 (1H, s, 2′-H), 4.26 (1H, s, 3′-H), 3.88 (1H, d, J 8.1,1″-H_(a)), 3.85-3.55 (7H, m, 1″-H_(b), OCH₃), 3.58-3.40 (2H, m,5′-H_(a), 5′-H_(b)). δ_(C) (CDCl₃) 164.10 (C-4), 158.60 (DMT), 150.45(C-2), 147.53 (DMT), 144.51 (C-6), 139.72, 135.49, 135.37, 130.20,129.28, 128.09, 127.85, 127.07 (DMT), 113.39, 113.17 (DMT), 101.79(C-5), 88.20, 87.10, 86.87 (C-1′, C4′, DMT), 79.25, 71.79, 69.70, 58.13(C-2′, C-3′, C-5′, C-1″), 55.33 (OCH₃). FAB-MS m/z 559 [M+H]⁺.

Example 48

(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)posphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(46). To a solution of compound 45 (0.07 g, 0.125 mmol) in anhydrousdichloromethane (2 cm³) at room temperature was addedN,N-diisopropylethylamine (0.1 cm³) and 2-cyanoethylN,N-diisopropylphosphoramidochloridite (0.07 cm³, 0.32 mmol). Afterstirring for 1 h, the reaction was quenched with MeOH (2 cm³), and theresulting mixture was diluted with ethyl acetate (5 cm³) and washedsuccessively with saturated aqueous solutions of sodiumhydrogencarbonate (3×2 cm³) and brine (3×2 cm³), and was dried (Na₂SO₄).The solvent was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give a white foam.This foam was dissolved in dichloromethane (2 cm³) and the product wasprecipitated from petroleum ether (10 cm³, cooled to −30° C.) undervigorous stiffing. The precipitate was collected by filtration and wasdried to give compound 46 as a white solid material (0.055 g, 58%).δ_(P) (CDCl₃) 149.18, 149.02.

Example 49

9-(2-O-Acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-(3-D-ribofuranosyl)-2-N-isobutyrylguanine(47). To a stirred suspension of the anomeric mixture 33 (1.28 g, 5.6mmol) and 2-N-isobutyrylguanine (1.8 g, 3.7 mmol) in anhydrousdichloroethane (60 cm³) was added N,O-bis(trimethylsilyl)acetamide (4cm³, 16.2 mmol). The reaction mixture was stirred at reflux for 1 h.Trimethylsilyl triflate (1.5 mL, 8.28 mmol) was added dropwise at 0° C.and the solution was stirred at reflux for 2 h. The reaction mixture wasallowed to cool to room temperature during 1.5 h. After dilution to 250cm³ by addition of dichloromethane, the mixture was washed with asaturated aqueous solution of sodium hydrogencarbonate (200 cm³) andwater (250 cm³). The solvent was removed under reduced pressure and theresidue was purified by silica gel column chromatography using 1.25%(200 cm³) and 1.5% (750 cm³) of methanol in dichloromethane (v/v) aseluents to give 2.10 g (87%) of a white solid that according to ¹H-NMRanalysis consisted of three isomers (ratio: 12.5:2.5:1). The mainproduct formed in that conditions is expected to be compound 47 (P.Garner, S. Ramakanth, J. Org. Chem. 1988, 53, 1294; H. Vorbruggen, K.Krolikiewicz, B. Bennua, Chem. Ber. 1981, 114, 1234). The individualisomers were not isolated and mixture was used for next step. For mainproduct 47: δ_(H) (CDCl₃) 12.25 (br s, NHCO), 9.25 (br s, NH), 7.91 (s,8-H) 7.39-7.26 (m, Bn), 6.07 (d, J 4.6, 1′-H), 5.80 (dd, J 5.8, J 4.7,2′-H), 4.72 (d, J 5.9, 3′-H), 4.59-4.43 (m, Bn, 1″-H_(a)), 4.16 (d, J12.1, 1″-H_(b)), 3.70 (d, J 10.1, 5′-H_(a)), 3.58 (d, J 10.1, 5′-H_(b)),2.65 (m, CHCO), 2.05 (s, COCH₃), 2.01 (s, COCH₃), 1.22 (d, J 6.7,CH₃CH), 1.20 (d, J 7.0, CH₃CH). (CDCl₃) 178.3 (COCH), 170.6, 179.8(COCH₃), 155.8, 148.2, 147.6 (guanine), 137.6, 137.2 (guanine, Bn),128.5, 128.4, 128.2, 128.1, 128.0, 127.8, 127.7 (Bn), 121.2 (guanine),86.2, 86.0 (C-1′, C-4′), 77.8 (C-3′), 74.9, 74.5, 73.7, 70.4 (Bn, C-2′,C-5′), 63.5 (C-1″), 36.3 (COCH), 20.8, 20.6 (COCH₃), 19.0 (CH₃CH). Forthe mixture: FAB-MS m/z 648 [M+H]⁺, 670 [M+Na]⁺. Found: C, 60.8; H, 6.0;N, 10.4; C₃₃H₃₆N₅O₉ requires C, 61.3; H, 5.6; N, 10.8%.

Example 50

9-(3,5-Di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl)-2-N-isobutyrylguanine(48). A solution of the mixture described in Example 49 containingcompound 47 (2.10 g, 3.25 mmol) in THF/Pyridine/methanol (2:3:4, v/v/v)(40 cm³) was cooled to −10° C. and sodium methoxide (320 mg, 5.93 mmol)was added to the stirred solution. The reaction mixture was stirred at10° C. for 30 min and neutralised with 2 cm³ of acetic acid. The solventwas evaporated under reduced pressure and the residue was twiceextracted in a system of dichloromethane/water (2×100 cm³). The organicfractions were combined and evaporated under reduced pressure. Afterco-evaporation with toluene, the residue was purified by silica gelcolumn chromatography in a gradient (2-7%) of methanol indichloromethane (v/v) to give a white solid material (1.62 g). Accordingto ¹H-NMR it consisted of three isomers (ratio: 13.5:1.5:1). For mainproduct 48: δ_(H) (CD₃OD) 8.07 (s, 8-H) 7.36-7.20 (m, Bn), 6.05 (d, J3.9, 1′-H), 4.81 (d, J 11.5, Bn), 4.75 (m, 2′-H), 4.56 (d, J 11.5, Bn),4.51-4.43 (m, Bn, 3′-H), 3.83 (d, J 11.7, 1″-H_(a)), 3.65 (d, J 11.7,1″-H_(b)), 3.64 (d, J 10.6, 5′-H_(a)), 3.57 (d, J 10.3, 5′-H_(b)), 2.69(m, CHCO), 1.20 (6H, d, J 6.8, CH₃CH). δ_(C) (CD₃OD) 181.6 (COCH),157.3, 150.2, 149.5 (guanine), 139.4, 139.3, 139.0 (guanine, Bn), 129.5,129.4, 129.3, 129.2, 129.1, 129.0, 128.9, 128.8 (Bn), 121.2 (guanine),90.7, 89.6 (C-1′, C-4′), 79.2 (C-3′), 75.8, 74.5, 74.3, 72.2 (Bn, C-2′,C-5′), 63.1 (C-1″), 36.9 (COCH), 19.4 (CH₃CH), 19.3 (CH₃CH). For themixture: FAB-MS m/z 564 [M+H]⁺.

Example 51

Intermediate 49. A solution of the mixture described in Example 50containing 48 (1.6 g) in anhydrous pyridine (6 cm³) was stirred at −20°C. and p-toluenesulphonyl chloride (0.81 g, 4.27 mmol) was added. Thesolution was stirred for 1 h at −20° C. and for 2 h at −25° C. Then themixture was diluted to 100 cm³ by addition of dichloromethane andimmediately washed with water (2×100 cm³). The organic phase wasseparated and evaporated under reduced pressure. The residue waspurified by silica gel column chromatography usingdichloromethane/methanol as eluent (1-2%, v/v) to give intermediate 49(980 mg). After elution of compound 49 from the column, the startingmixture containing 48 (510 mg) was eluted using 8% methanol indichloromethane (v/v) as eluent. This material was concentrated, driedunder reduced pressure and treated in the same manner as described aboveto give additionally 252 mg of the intermediate. The intermediate (1.23g) was purified by silica gel HPLC (PrepPak Cartridge packed by Porasil,15-20 μm, 125A, flow rate 60 cm³/min, eluent 0-4% of methanol indichloromethane (v/v), 120 min). Fractions containing intermediate 49were pooled and concentrated to give white solid (1.04 g). According to¹H-NMR it consisted of two main products, namely 1″-O and 2′-Omonotosylated derivatives in a ratio of ˜2:1. FAB-MS m/z 718 [M+H]⁺.Found C, 60.4; H, 5.8; N, 9.3; C₃₆H₃₉N₅O₉S requires C, 60.2; H, 5.5; N,9.8%.

Example 52

(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(2-N-isobutyrylguanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(50). To a solution of intermediate 49 (940 mg) in anhydrous THF (20cm³) was added a 60% suspension of sodium hydride (w/w, 130 mg, 3.25mmol) and the mixture was stirred for 1 h at room temperature. Aceticacid (0.25 mL) was added and the mixture was concentrated under reducedpressure. The residue was dissolved in dichloromethane (100 cm³) and waswashed with water (2×100 cm³). The organic phase was separated andevaporated under reduced pressure. The residue was purified by silicagel column chromatography using methanol/dichloromethane (1-1.5%, v/v)as eluent to give nucleoside 50 as a white solid material (451 mg, 57%).δ_(H) (CDCl₃) 12.25 (1H, br s, NHCO), 10.12 (1H, br s, NH), 7.84 (1H, s,8-H), 7.31-7.15 (10H, m, Bn), 5.72 (1H, s, 1′-H), 4.60-4.46 (5H, m, Bn,2′-H), 4.14 (1H, s, 3′-H), 4.02 (1H, d, J 7.9, 1″-H_(a)), 3.85 (1H, d, J7.9, 1″-H_(b)), 3.78 (2H, s, 5′-H), 2.81 (1H, m, CHCO), 1.24 (3H, d, J6.8, CH₃CH), 1.22 (3H, d, J 6.4, CH₃CH). (CDCl₃) 179.5 (COCH), 155.6,148.1, 147.3 (guanine), 137.3, 136.9, 136.0 (guanine, Bn), 128.4, 128.3,127.9, 127.8, 127.5, 127.4 (Bn), 121.2 (guanine), 87.1, 86.2 (C-1′,C-4′), 77.0 (C-3′), 73.6, 72.5, 72.1 (Bn, C-2′, C-5′), 64.9 (C-1″), 36.1(COCH), 19.0 (CH₃CH), 18.9 (CH₃CH). FAB-MS m/z 546 [M+H]⁺. Found: C,63.3; H, 5.9; N, 12.5; C₂₉H₃₀N₅O₆ requires C, 64.0; H, 5.6; N, 12.9%.

Alternative preparation of compound 50. G1AQ. To a suspension ofcompound 78 (1.5 g, 2.51 mmol), N2-isobutirylguanine (0.93 g, 4.06 mmol)in dry DCM (50 mL) was added BSA (N,O-bistrimethylsilylacetamide; 3.33mL, 13.5 mmol) and the mixture was refluxed for 2 h. Trimethylsilyltriflate (1.25 mL, 6.9 mmol) was added to the mixture and refluxing wascontinuing for additional 2 h. The mixture was allowed to cool to roomtemperature, diluted by 200 mL of DCM and washed by saturated aq. NaHCO₃and water. Chromatography at silica gel column (1-2.5% of CH₃OH indichloromethane) yielded 1.05 g (55%) of the desired isomer G1AQ and 380mg of isomers with higher mobility which was converted to G1AQ byrepetition of the procedure described above. Ammonium hydroxide (12 mLof 25% aq. solution) was added to a solution of G1AQ (1.05 g in 12 mL ofmethanol) and the mixture was stirred for 1 hr at room temperature.After concentration, the product was purified by silica gel columnchromatography (1-3% CH₃OH in dichloromethane) to give 700 mg G3 as awhite solid material. 700 mg of G3 in anhydroux THF (15 mL) was treatedwith NaH (225 mg of 60% suspension in mineral oil). 30 min later, thereaction was quenched by addition of 1.25 mL of acetic acid, and themixture was concentrated under reduced pressure. The residue wasdissolved in dichloromethane, washed by NaHCO₃ and water and purified bysilica gel chromatography in gradient 0.5-3% methanol/DCM. Yield 400 mg(75%) of 50.

Example 53

(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(2-N-isobutyrylguanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(51). A mixture of nucleoside 50 (717 mg, 1.31 mmol) and 10% palladiumover carbon (500 mg) was suspended in methanol (8 cm³) at roomtemperature. The mixture was degassed several times under reducedpressure and placed under a hydrogen atmosphere. After stirring for 24 hthe mixture was purified by silica gel column chromatography usingmethanol/dichloromethane (8-20%, v/v) as eluent to give nucleoside 51 asa glass-like solid (440 mg, 92%). δ_(H) (CD₃OD) 8.12 (1H, br s, 8-H),5.86 (1H, s, 1′-H), 4.50 (1H, s, 2′-H), 4.30 (1H, s, 3′-H), 4.05 (1H, d,J 8.0, 1″-H_(a)), 3.95 (2H, s, 5′-H), 3.87 (1H, d, J 7.9, 1″-H_(b)),2.74 (1H, m, CHCO), 1.23 (6H, d, J 6.9, CH₃CH). (CD₃OD, signals from thecarbohydrate part) 90.2, 87.6 (C-1′, C-4′), 81.1 (C-3′), 72.9, 71.3(C-2′, C-5′), 58.2 (C-1″), 37.1 (COCH), 19.5 (CH₃CH). FAB-MS m/z 366[M+H]⁺.

Example 54

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-(2-N-isobutyrylguanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(52). A mixture of compound 51 (440 mg, 1.21 mmol) and4,4′-dimethoxytrityl chloride (573 mg, 1.69 mmol) was dissolved inanhydrous pyridine (7 cm³) and was stirred at room temperature for 4 h.The mixture was evaporated under reduced pressure to give an oil.Extraction was performed in a system of dichloromethane/water (1:1, v/v,40 cm³). The organic phase was separated and concentrated to give asolution in a minimal volume of dichloromethane containing 0.5% ofpyridine (v/v) which was applied to a silica gel column equilibrated bythe same solvent. The product was eluted in gradient concentrations ofmethanol (0.6-2%, v/v) in dichloromethane containing 0.5% of pyridine(v/v) to give compound 52 as a white solid material (695 mg, 86%). δ_(H)(CDCl₃) 12.17 (1H, br s, NHCO), 10.09 (1H, br s, NH), 7.87 (1H, s, 8-H),7.42-6.72 (13H, m, DMT), 5.69 (1H, s, 1′-H), 4.59 (1H, s, 2′-H), 4.50(1H, s, 3′-H), 3.98 (1H, d, J 8.1, 1″-H_(a)), 3.69-3.39 (9H, m, DMT,5′-H, 1″-H_(b)), 2.72 (1H, m, CHCO), 1.17 (6H, d, J 6.8, CH₃CH). (CDCl₃)179.8 (COCH), 158.8, 144.5, 135.6, 135.5, 130.1, 128.1, 127.7, 126.9,113.2 (DMT), 155.8, 147.9, 147.5, 137.0, 120.8 (guanine), 87.6, 86.4,86.1 (C-1′, C-4′, DMT), 79.7 (C-3′), 72.6, 71.4 (C-2′, C-5′), 59.8(C-1″), 55.2 (DMT), 36.1 (COCH), 19.1, 18.8 (CH₃CH). FAB-MS m/z 668[M+H]⁺.

Example 55

(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(2-N-isopropyonylguanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(53). Compound 52 (670 mg, 1.0 mmol) was at room temperature dissolvedin anhydrous dichloromethane (5 cm³) containingN,N-diisopropylethylamine (0.38 cm³, 4 mmol). 2-CyanoethylN,N-diisopropylphosphoramidochloridite (0.36 cm³, 2.0 mmol) was addeddrop-wise with stirring. After 5 h, methanol (2 cm³) was added and themixture was diluted to 100 cm³ by addition of dichloromethane and washedwith a saturated aqueous solution of sodium hydrocarbonate (50 cm³). Theorganic phase was separated and the solvent was removed by evaporationunder reduced pressure. The residue was dissolved in the minimun amountof dichloromethane/petroleum ether (1:1, v/v) containing 0.5% pyridine(v/v) and was applied to a column packed with silica gel equilibrated bythe same solvent mixture. The column was washed bydichloromethane/petroleum/pyridine (75:25:0.5, v/v/v, 250 cm³) and theproduct was eluted using a gradient of methanol in dichloromethane(0-1%, v/v) containing 0.5% pyridine (v/v). The fractions containing themain product were evaporated and co-evaporated with toluene. The residuewas dissolved in anhydrous dichloromethane (5 cm³) and precipitated frompetroleum ether (100 cm³) to give compound 53 as a white solid material(558 mg, 64%) after filtration and drying. δ_(P) (CDCl₃) 148.17, 146.07.FAB-MS m/z 868 [M+1]⁺.

Example 56

1-(2-O-Acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-β-D-ribofuranosyl)-4-N-benzoylcytosine(54). To a stirred solution of the anomeric mixture 33 (4.0 g, 8.22mmol) and 4-N-benzoylcytosine (2.79 g, 13.0 mmol) was addedN,O-bis(trimethylsilyl)acetamide (8.16 cm³, 33.0 mmol). The reactionmixture was stirred for 1 h at room temperature and cooled to 0° C.Trimethylsilyl triflate (3.0 cm³, 16.2 mmol) was added dropwise and themixture was stirred at 60° C. for 2 h. Saturated aqueous solutions ofsodium hydrogencarbonate (3×20 cm³) and brine (2×20 cm³) weresuccessively added, and the separated organic phase was dried (Na₂SO₄).The solvent was removed under reduced pressure and the residue waspurified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give compound 54 as awhite solid material (3.9 g, 74%). δ_(H) (CDCl₃), 8.28 (1H, d, J 7.5,6-H), 7.94-7.90 (2H, m, Bz), 7.65-7.25 (13H, m, Bn, Bz), 7.16 (1H, d, J7.1, 5-H), 6.22 (1H, d, J 2.8, 1′-H), 5.51 (1H, dd, J 2.8, 5.8, 2′-H),4.62 (1H, d, J 11.6, Bn), 4.51 (1H, d, J 12.3, 1″—H_(a)), 4.49-4.34 (4H,m, 3′-H, Bn), 4.21 (1H, d, J 12.3, 1″-H_(b)), 3.85 (1H, d, J 10.3,5′-H_(a)), 3.47 (1H, d, J 10.3, 5′-H_(b)), 2.13 (3H, s, COCH₃), 2.06(3H, s, COCH₃). δ_(C) (CDCl₃) 170.52, 169.61 (C═O), 166.83, 162.27 (C-4,C═O), 154.26 (C-2), 145.26 (C-6), 137.25, 136.93, 133.18, 129.0, 128.75,128.51, 128.45, 128.18, 128.10, 127.89, 127.71 (Bn, Bz), 96.58 (C-5),89.42, 86.52 (C-1′, C-4′), 76.21, 75.10, 74.17, 73.70, 69.70, 63.97(C-2′, C-3′, Bn, C-5′, C-1″), 20.82 (COCH₃). FAB-MS m/z 664 [M+Na]⁺, 642[M+H]⁺. Found: C, 65.0; H, 5.7; N, 6.5; C₃₅H₃₅N₃O₉ requires C, 65.5; H,5.5; N, 6.5%.

Example 57

1-(3,5-Di-O-benzyl-4-C-hydroxymethyl-(3-D-ribofuranosyl)-4-N-benzoylcytosine(55). To a stirred solution of nucleoside 54 (3.4 g, 5.3 mmol) inmethanol (20 cm³) was added sodium methoxide (0.663 g, 11.66 mmol). Thereaction mixture was stirred at room temperature for 10 min and thenneutralised with 20% aqueous hydrochloric acid. The solvent was partlyevaporated and the residue was extracted with dichloromethane (3×50cm³). The combined organic phase was washed with a saturated aqueoussolution of sodium hydrogencarbonate (3×20 cm³) and was dried (Na₂SO₄).The solvent was removed under reduced pressure and the residue waspurified by silica gel column chromatography usingdichloromethane/methanol (98.5:1.5, v/v) as eluent to give compound 55as a white solid material (1.6 g, 54%). δ_(H) (CDCl₃) 9.95 (1H, br s,NH), 8.33 (1H, d, J 7.4, 6-H), 7.98 (2H, m, Bz), 7.60-7.12 (14H, m, Bn,Bz, 5-H), 6.17 (1H, d, J1.6, 1′-H), 4.78 (1H, d, J 11.8, Bn), 4.48-4.27(5H, m, Bn, 2′-H, 3′-H), 3.85 (1H, d, J 11.8, 5′-H_(a)), 3.66-3.61 (2H,m, 5′-H_(b), 1″-H_(a)), 3.47 (1H, d, J 10.4, 1″-H_(b)). δ_(C) (CDCl₃)167.5, 162.31 (C-4, C═O), 155.36 (C-2), 145.34 (C-6), 137.49, 137.08,133.09, 133.01, 128.94, 128.67, 128.48, 128.30, 128.01, 127.90, 127.80(Bn, Bz), 96.53 (C-5), 93.97, 89.35 (C-1′, C-4′), 76.06, 75.28, 73.70,72.76, 70.26, 62.44 (C-2′, C-3′, Bn, C-5′, C-1″). FAB-MS m/z 558 [M+H]⁺.

Example 58

Intermediate 56. A solution of nucleoside 55 (2.2 g, 3.94 mmol) inanhydrous tetrahydrofuran (60 cm³) was stirred at −20° C. and asuspension of 60% sodium hydride in mineral oil (w/w, 0.252 g, 6.30mmol) was added in seven portions during 45 min. The solution wasstirred for 15 min at −20° C. followed by addition of p-toluenesulfonylchloride (0.901 g, 4.73 mmol) in small portions. The solution wasstirred for 4 h at −20° C. Additional sodium hydride (0.252 g, 6.30mmol) and p-toluenesulfonyl chloride (0.751 g, 3.93 mmol) was added. Thereaction mixture was kept at −20° C. for 48 h. The reaction was quenchedby addition of ice-cold water (50 cm³) whereupon extraction wasperformed with dichloromethane (3×60 cm³). The combined organic phasewas washed with a saturated aqueous solution of sodium hydrogencarbonate(3×20 cm³) and dried (Na₂SO₄). The solvent was evaporated under reducedpressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (99:1, v/v) as eluent togive the intermediate 56 (1.80 g).

Example 59

(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(4-N-benzoylcytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(57). Intermediate 56 (1.80 g) was dissolved in anhydrous DMF (30.0 cm³)and a 60% suspension of sodium hydride in mineral oil (w/w, 0.16 g, 3.9mmol) was added in five portions during 30 min at 0° C. The reactionmixture was stirred for 36 h at room temperature. The reaction wasquenched by adding ice-cold water (70 cm³) and the resulting mixture wasextracted with dichloromethane (3×50 cm³). The combined organic phasewas washed with a saturated aqueous solution of sodium hydrogencarbonate(3×30 cm³) and dried (Na₂SO₄). The solvents were removed under reducedpressure and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (99.5:0.5, v/v) as eluentto give compound 57 as a white solid material (1.08 g, 79%). δ_(H)(CDCl₃) 8.95 (1H, br s, NH), 8.20 (1H, d, J 7.5, 6-H), 7.95-7.92 (2H, m,Bz), 7.66-7.22 (14H, m, Bn, Bz, 5-H), 5.78 (1H, s, 1′-H), 4.70-4.65 (3H,m, 2′-H, Bn), 4.60 (1H, d, J 11.6, Bn), 4.47 (1H, d, J 11.6, Bn),4.05-3.78 (5H, m, 3′-H, 5′-H_(a), 1″-H_(a), 5′-H_(b), 1″-H_(b)). δ_(C)(CDCl₃) 167.0, 162.36 (C-4, C═O), 154.5 (C-2), 144.58 (C-6), 137.46,136.93, 133.35, 132.93, 129.11, 128.67, 128.50, 128.16, 128.11, 127.68,127.60 (Bn), 96.35 (C-5), 88.38, 87.67 (C-1′, C-4′), 76.14, 75.70,73.79, 72.27, 72.09, 64.34 (Bn, C-5′, C-1″, C-2′, C-3′). FAB-MS m/z 540[M+H]⁺. Found: C, 68.0; H, 5.5; N, 7.5; C₃₁H₂₉N₃O₆ requires C, 69.0; H,5.4; N, 7.8%).

Example 60

(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(cytosin-1-yl)-2,5-dioxabicyclo-[2.2.1]heptane(57A). To a solution of nucleoside 57 (0.3 g, 0.55 mmol) in anhydrousmethanol (22 cm³) were added 1,4-cyclohexadiene (5.0 cm³) and 10%palladium on carbon (0.314 g). The mixture was stirred under reflux for18 h. Additional 10% palladium on carbon (0.380 g) and1,4-cyclohexadiene (5.5 cm³) were added and the mixture was refluxed for54 h. The reaction mixture was filtered through a pad of silica gelwhich was subsequently washed with methanol (1500 cm³). The combinedfiltrate was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography usingdichloromethane/methanol (92.5:7.5, v/v) as eluent to give compound 57Aas a white solid material (0.051 g, 36%). δ_(H) ((CD₃)₂SO) 7.73 (1H, d,J 7.7, 6-H), 7.12-7.20 (2H, br s, NH₂), 5.74 (1H, d, J7.7, 5-H), 5.61(1H, br s, 3′-OH), 5.39 (1H, s, 1′-H), 5.12 (1H, m, 5′-OH), 4.08 (1H, s,2′-H), 3.80 (1H, d, J 7.7, 1″-H_(a)), 3.81 (1H, s, 3′-H), 3.74 (2H, m,5′-H_(a), 5′-H_(b)), 3.63 (1H, d, J7.7, 1″-H_(b)). δ_(C) ((CD₃)₂SO)165.66 (C-4), 154.58 (C-2), 139.68 (C-6), 93.19 (C-5), 88.42, 86.73(C-1′, C-4′), 78.87, 70.85, 68.32, 56.04 (C-2′, C-1″, C-3′, C-5′).FAB-MS m/z 256 [M+H]⁺.

Example 61

Intermediate 57B. To nucleoside 57A (0.030 g, 0.11 mmol) suspended inanhydrous pyridine (2.0 cm³) was added trimethylsilyl chloride (0.14cm³, 1.17 mmol) and stiffing was continued for 1 h at room temperature.Benzoyl chloride (0.07 cm³, 0.58 mmol) was added at 0° C. and themixture was stirred for 2 h at room temperature. After cooling thereaction mixture to 0° C., water (3.0 cm³) was added. After stirring for5 min, an aqueous solution of ammonia (1.5 cm³, 32%, w/w) was added andstirring was continued for 30 min at room temperature. The mixture wasevaporated under reduced pressure, and the residue was purified bysilica gel column chromatography using dichloromethane/methanol(97.5:2.5, v/v) as eluent to give intermediate 57B as white solidmaterial (0.062 g).

Example 62

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-(4-N-benzoylcytosine-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(57C). To a solution of intermediate 57B (0.042 g, 0.11 mmol) inanhydrous pyridine (1.5 cm³) was added 4,4′-dimethoxytrityl chloride(0.06 g, 0.17 mmol). The reaction mixture was stirred at roomtemperature for 3.5 h, cooled to 0° C., and a saturated aqueous solutionof sodium hydrogencarbonate (20 cm³) was added. Extraction was performedusing dichloromethane (3×10 cm³). The combined organic phase was dried(Na₂SO₄) and evaporated to dryness under reduced pressure. The residuewas purified by silica gel column chromatography usingdichloromethane/methanol/pyridine (98.0:1.5:0.5, v/v/v) as eluent togive nucleoside 57C as a white solid material (0.031 g, ˜63% from 57A).δ_(H) (C₅D₅N) 12.32 (1H, br s, NHCO), 8.75-7.06 (20H, m, DMT, Bz, H-5,H-6), 6.24 (1H, s, 1′-H), 5.11 (1-H, s, 2′-H), 4.90 (1H, s, 3′-H), 4.38(1H, d, J 7.6, 1″-H_(a)), 4.10 (1H, d, J 7.6, 1″-H_(b)), 4.02 (1H, d, J10.6, 5′-H_(a)), 3.87 (1H, d, J 10.6, 5′-H_(b)), 3.77, 3.76 (2×3H, 2×s,2×OCH₃). δ_(C) (C₅D₅N) 169.00 (NHCO), 164.24 (C-2), 159.39 (DMT), 150.5,145.62 (DMT), 144.31, 132.89, 130.82, 130.72, 129.09, 128.89, 128.60,113.96 (DMT), 96.96, 89.01, 87.18, 79.91, 72.56, 70.25 (C-5, C-1′, C-4′,C-2′, C-1″, C-3′), 59.51 (C-5′), 55.33 (OCH₃). FAB-MS m/z 662 [M+H]⁺.

Example 63

(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(4-N-benzoylcytosine-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(57D). To a solution of nucleoside 57C (0.025 g, 0.03 mmol) in anhydrousdichloromethane (1.5 cm³) was added N,N-diisopropylethylamine (0.03 cm³,0.17 mmol) followed by dropwise addition of 2-cyanoethylN,N-diisopropylphosphoramidochloridite (0.02 cm³, 0.09 mmol). Afterstirring for 5 h at room temperature, the reaction mixture was cooled to0° C., dichloromethane/pyridine (10.0 cm³, 99.5:0.5, v/v) was added, andwashing was performed using a saturated aqueous solution of sodiumhydrogencarbonate (3×8 cm³). The organic phase was separated, dried(Na₂SO₄) and evaporated to dryness under reduced pressure. The residuewas purified by silica gel column chromatography usingdichloromethane/methanol/pyridine (99.0:0.5:0.5, v/v/v) as eluent togive amidite 57D as a light yellow oil (0.038 g). δ_(P) (CDCl₃) 147.93.

Example 64

9-(2-O-Acetyl-4-C-acetyloxymethyl-3,5-di-O-benzyl-β-D-ribofuranosyl)-6-N-benzoyladenine(58). To a stirred suspension of the anomeric mixture 33 (5.0 g, 10.3mmol) and 6-N-benzoyladenine (3.76 g, 15.7 mmol) in anhydrousdichloromethane (200 cm³) was added N,O-bis(trimethylsilyl)acetamide(15.54 cm³, 61.8 mmol). The reaction mixture was stirred at reflux for 1h and then cooled to room temperature. Trimethylsilyl triflate (7.0 cm³,38.7 mmol) was added dropwise and the mixture was refluxed for 20 h. Thereaction mixture was allowed to cool to room temperature and the volumeof the mixture was reduced to ¼ under reduced pressure. Dichloromethane(250 cm³) was added, and the solution was washed with a saturatedaqueous solution of sodium hydrogencarbonate (3×50 cm³) and water (50cm³). The organic phase was dried (Na₂SO₄) and evaporated under reducedpressure. The residue was purified by silica gel column chromatographyusing dichloromethane/methanol (99.5:0.5, v/v) as eluent to givenucleoside 58 as white solid material (3.65 g, 52%). δ_(H) (CDCl₃) 9.25(1H, br s, NH), 8.71 (1H, s, 8-H), 8.24 (1H, s, 2-H), 8.0 (2H, d, J 7.5,Bz), 7.60-7.23 (13H, m, Bn, Bz), 6.35 (1H, d, J 4.6, 1′-H), 5.99 (1H,dd, J 4.9, 5.3, 2′-H), 4.78 (1H, d, J 5.6, 3′-H), 4.64-4.42 (5H, m, Bn,1″-H_(a)), 4.25 (1H, d, J 12.1, 1″-H_(b)), 3.72 (1H, d, J 10.1,5′-H_(a)), 3.56 (1H, d, J 10.1, 5′-H_(b)), 2.07 (3H, s, COCH₃), 2.02(3H, s, COCH₃). δ_(C) (CDCl₃) 170.42, 169.72 (COCH₃), 164.60 (NHCO),152.51 (C-6), 151.45 (C-2), 149.46 (C-4), 141.88 (C-8), 137.04, 137.00,133.50, 132.60, 128.86, 128.66, 128.53, 128.41, 128.38, 128.18, 128.06,127.91, 127.88, 127.79, 127.63, 123.26 (Bz, Bn, C-5), 86.38 (C-1′),86.25 (C-4′), 77.74, 74.74, 74.44, 73.48 (C-2′, C-3′, 2×Bn), 70.11(C-1″), 63.42 (C-5′), 20.70, 20.54 (COCH₃). FAB-MS m/z 666 [M+H]⁺.

Example 65

9-(3,5-Di-O-benzyl-4-C-hydroxymethyl-β-D-ribofuranosyl)-6-N-benzoyladenine(59). To a stirred solution of nucleoside 58 (4.18 g, 6.28 mmol) inmethanol (50 cm³) was added sodium methoxide (0.75 g, 13.8 mmol) at 0°C. The reaction mixture was stirred for 2 h, and ice was added. Themixture was neutralised using a 20% aqueous solution of HCl. Extractionwas performed using dichloromethane (3×75 cm³), the organic phase wasseparated, dried (Na₂SO₄) and evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography usingdichloromethane/methanol (98.5:1.5, v/v) as eluent to give nucleoside 59as a white solid material (2.68 g, 73%). δ_(H) (CDCl₃) 9.42 (1H, br s,NH), 8.58 (1H, s, H-8), 8.16 (1H, s, 2-H), 7.96 (2H, d, J 7.2, Bz),7.52-7.08 (13H, m, Bn, Bz), 6.18 (1H, d, J 2.5, 1′-H), 4.85-4.38 (4H, m,Bn, 2′-H, 3′-H), 4.33 (2H, s, Bn) 3.90 (1H, d, J 11.9, 1″-H_(a)), 3.71(1H, d, J 11.8, 1″-H_(b)), 3.50-3.39 (2H, m, 5-H). δ_(C) (CDCl₃) 164.98(NHCO), 152.19 (C-6), 151.00 (C-2), 149.34 (C-4), 142.28 (C-8), 137.32,137.25, 133.46, 132.70, 128.69, 128.49, 128.40, 128.11, 128.03, 127.94,127.83, 127.62, (Bz, Bn), 122.92 (C-5), 90.94, 88.75 (C-1′, C-4′),77.65, 74.08, 73.44, 73.20, 71.12, 62.39 (C-1″, C-5′, C-2′, C-3′, 2×Bn).FAB-MS m/z 582 [M+H]⁺. Found: C, 65.6; H, 5.5; N, 11.7; C₃₂H₃₁N₅O₆requires C, 66.1; H, 5.4; N, 12.0%.

Example 66

Intermediate 60. A solution of nucleoside 59 (2.43 g, 4.18 mmol) inanhydrous tetrahydrofuran (25 cm³) was stirred at −20° C. and a 60%suspension of sodium hydride in mineral oil (w/w, 0.28 g, 7.0 mmol) wasadded in four portions during 30 min. After stirring for 1 h,p-toluenesulfonyl chloride (1.34 g, 7.0 mmol) was added in smallportions. The mixture was stirred at −10° C. for 15 h. Ice-cold water(50 cm³) was added and extraction was performed with dichloromethane(3×50 cm³). The combined organic phase was washed with a saturatedaqueous solution of sodium hydrogencarbonate (2×25 cm³), dried (Na₂SO₄)and evaporated under reduced pressure. The residue was purified bysilica gel column chromatography using dichloromethane/methanol (99:1,v/v) as eluent to give the intermediate 60 (1.95 g).

Example 67

(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(61). Intermediate 60 (1.90 g) was dissolved in anhydrous DMF (20 cm³)and a 60% suspension of sodium hydride in mineral oil (w/w, 0.16 g, 3.87mmol) was added in small portions at 0° C. The mixture was stirred for10 h at room temperature and then concentrated under reduced pressure.The residue was dissolved in dichloromethane (75 cm³), washed with asaturated aqueous solution of sodium hydrogencarbonate (2×25 cm³), dried(Na₂SO₄), and evaporated under reduced pressure. The residue waspurified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give nucleoside 61 aswhite solid material (1.0 g, ˜44% from 59). δ_(H) (CDCl₃) 8.71 (H, s,8-H), 8.23 (1H, s, 2-H), 8.02 (2H, m, J 7.0, Bz), 7.99-7.19 (13H, m, Bn,Bz), 6.08 (1H, s, 1′-H), 4.78 (1H, s, 2′-H), 4.61-4.50 (4H, m, 2×Bn),4.24 (1H, s, 3′-H), 4.12 (1H, d, J 7.8, 1″-H_(a)), 4.00 (1H, d, J 7.9,1″-H_(b)), 3.85-3.78 (2H, m, 5′-H_(a), 5′-H_(b)). δ_(C) (CDCl₃) 164.61(NHCO), 152.32 (C-6), 150.61 (C-2), 149.35 (C-4), 140.67 (C-8), 137.24,136.76, 133.33, 132.66, 128.68, 128.39, 128.29, 127.94, 127.77, 127.51(Bn, Bz), 123.43 (C-5), 87.14, 86.52 (C-1′, C-4′), 77.21, 76.77, 73.56,72.57, 72.27, 64.65 (C-2′, C-3′, C-1″, 2×Bn, C-5′). FAB-MS m/z 564[M+H]⁺. Found: C, 66.2; H, 5.5; N, 11.4; C₃₂H₂₉N₅O₅ requires C, 66.2; H,5.2; N, 12.4%.

Example 68

(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(adenin-9-yl)-2,5-dioxabicyclo-[2.2.1]heptane(61A). To a stirred solution of nucleoside 61 (0.80 g, 1.42 mmol) inanhydrous dichloromethane (30 cm³) at −78° C. was dropwise during 30 minadded BCl₃ (1 M solution in hexane; 11.36 cm³, 11.36 mmol). The mixturewas stirred for 4 h at −78° C., additional BCl₃ (1M solution in hexane,16.0 cm³, 16.0 mmol) was added drop-wise, and the mixture was stirred at−78° C. for 3 h. Then the temperature of the reaction mixture was raisedslowly to room temperature and stirring was continued for 30 min.Methanol (25.0 cm³) was added at −78° C., and the mixture was stirred atroom temperature for 12 h. The mixture was evaporated under reducedpressure, and the residue was purified by silica gel columnchromatography using dichloromethane/methanol (92:8, v/v) as eluent togive nucleoside 61A as a white solid material (0.332 g, 84%). δ_(H)((CD₃)₂SO) 8.22 (1H, s, 8-H), 8.15 (1H, s, 2-H), 7.33 (2H, s, NH₂), 5.89(1H, s, 1′-H), 5.83 (1H, d, J 4.2, 3′-OH), 5.14 (1H, t, J 5.9, 5′-OH),4.14 (1H, s, 2′-H), 4.25 (1H, d, J 4.2, 3′-H), 3.92 (1H, d, J 7.8,1″-H_(a)), 3.81-3.41 (3H, m, 5′-H_(a), 5′-H_(b), 1″-H_(b)). δ_(C)((CD₃)₂SO) 155.90 (C-6), 152.64 (C-2), 148.35 (C-4), 137.72 (C-8),118.94 (C-5), 88.48, 85.17 (C-1′, C-4′), 79.09, 71.34, 69.83, 56.51(C-2′, C-3′, C-1″, C-5′). FAB-MS m/z 280 [M+H]⁺.

Example 69

(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(61B). To a stirred solution of nucleoside 61A (0.32 g, 1.15 mmol) inanhydrous pyridine (1 cm³) was added trimethylsilyl chloride (0.73 cm³,5.73 mmol) and the mixture was stirred at room temperature for 20 min.Benzoyl chloride (0.67 cm³, 5.73 mmol) was added at 0° C., and thereaction mixture was stirred at room temperature for 2 h. The reactionmixture was cooled to 0° C. and ice-cold water (15.0 cm³) was added.After stiffing for 5 min, a 32% (w/w) aqueous solution of ammonia (1.5cm³) was added and the mixture was stirred for 30 min. The mixture wasevaporated to dryness and the residue was dissolved in water (25 cm³).After evaporation of the mixture under reduced pressure, the residue waspurified by silica gel chromatography using dichloromethane/methanol(97:3, v/v) as eluent to give nucleoside 61B as a white solid material(0.55 g). FAB-MS m/z 384 [M+H]⁺.

Example 70

(1R,3R,4R,7S)-7-Hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(61C). To a stirred solution of compound 61B (0.50 g) in anhydrouspyridine (20 cm³) was added 4,4′-dimethoxytrityl chloride (0.71 g, 2.09mmol) and 4-N,N-dimethylaminopyridine (DMAP) (0.1 g). After stiffing for2 h at room temperature and for 1 h at 50° C., the reaction mixture wascooled to 0° C. and a saturated aqueous solution of sodiumhydrogencarbonate (100 cm³) was added. After extraction usingdichloromethane (3×50 cm³), the combined organic phase was dried(Na₂SO₄) and evaporated under reduced pressure. The residue was purifiedby silica gel column chromatography eluting withdichloromethane/methanol/pyridine (98.0:1.5:0.5) to give nucleoside 61Cas a white solid material (0.36 g, ˜50% from 61A). δ_(H) (C₅D₅N) 12.52(NHCO), 9.10 (2H, d, J 7.7, Bz), 8.88 (1H, s, 8-H), 8.50-7.11 (17H, m,DMT, Bz, 2-H), 6.65 (1H, s, H-1′), 5.25 (2H, s, H-2′, H-3′), 4.71 (1H,d, J 7.8, 1″-H_(a)), 4.56 (1H, d, J 7.8, 1″-H_(b)), 4.20 (1H, d, J 10.8,5′-H_(a)), 4.07 (1H, d, J 10.8, 5′-H_(b)), 3.82, 3.81 (2×3H, 2×s,2×OCH₃). δ_(C) (C₅D₅N) 167.56 (NHCO), 159.24 (C-6), 152.50, 152.08,151.81, 145.84, 141.45, 136.52, 136.28, 132.55, 130.76, 130.70, 129.32,128.85, 128.76, 128.46, 127.38, 126.33 (DMT, Bz, C-2, C-4, C-8), 113.84(C-5), 88.64, 87.20, 86.85, 80.52, 73.13, 72.16, 60.86 (C-1′, C-4′, DMT,C-2′, C-3′, C-1″, C-5′), 55.24 (OCH₃). FAB-MS m/z 686 [M+H]⁺. Found: C,68.3; H, 5.0; N, 9.7; C₃₉H₃₅N₅O₇ requires C, 68.3; H, 5.1; N, 10.2%).

Example 71

(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(61D). To a solution of compound 61C (190 mg, 0.277 mmol) in anhydrousdichloromethane (1.5 cm³) were added N,N-diisopropylethylamine (0.16cm³, 0.94 mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite(0.1 cm³, 0.42 mmol) at 0° C. The mixture was allowed to warm to roomtemperature and stirred for 5 h. The solution was diluted bydichloromethane (50 cm³), washed by a saturated aqueous solution ofsodium hydrogencarbonate (2×30 cm³) and evaporated under reducedpressure. The products were isolated by silica gel HPLC (PrepPakcartridge, 25×100 mm, packed by Prep Nova-Pak® HR Silica 6 μm 60 ∪;gradient of solution B in solution A (from 0% to 15% during 25 min andfrom 15% to 100% during another 25 min, solution A:petroleum/dichloromethane/pyridine, 60/39.6/0.4, v/v/v, solution B:ethylacetate. The fractions containing the two main products (retentiontimes 30-40 min) were joined, concentrated under reduced pressure,co-evaporated with anhydrous toluene (2×40 cm³) and dried overnight invacuo. The residue was dissolved in anhydrous dichloromethane (4 cm³)and precipitated by adding this solution to anhydrous petroleum ether(80 cm³) under intensive stiffing. The precipitate was collected byfiltration, washed by petroleum ether (2×20 cm³) and dried under reducedpressure to give compound 61D (178 mg, 73%) as a white solid material.6_(p) (CD₃CN) 148.42, 147.93.

Example 72

1-(2,3-O-isopropylidene-4-C-(4-toluenesulphonyloxymethyl)-β-D-ribofura-nosyl)uridine(62). To a stirred solution of1-(2,3-O-isopropylidene-4′-C-hydroxymethyl-β-D-ribofuranosyl)uridine(2.0 g, 6.4 mmol) (R. Youssefyeh, D. Tegg, J. P. H. Verheyden, G. H.Jones and J. G. Moffat, Tetrahedron Lett., 1977, 5, 435; G. H. Jones, M.Taniguchi, D. Tegg and J. G. Moffat, J. Org. Chem., 1979, 44, 1309) inanhydrous pyridine (28 cm³) was added p-toluenesulfonyl chloride (1.46g, 7.3 mmol) dissolved in anhydrous pyridine (10 cm³) at −30° C. After30 min, the reaction mixture was allowed to reach room temperature andstiffing was continued at room temperature for 12 h. The reaction wasquenched with methanol (4 cm³), silica gel (2 g) was added and thesolvent was removed under reduced pressure. The residue was purified bysilica gel column chromatography using a gradient of 0-3% methanol indichloromethane (v/v) as eluent to give nucleoside 62 as a pale reddishsolid material (1.8 g, 60%). δ_(H) (CDCl₃) 9.80 (1H, br s, NH), 7.80(2H, d, J 8.3, Ts), 7.46 (1H, d, J 8.1, 6-H), 7.35 (2H, d, J 8.01, Ts),5.72 (1H, d, J 8.0, 5-H), 5.54 (1H, d, J 3.5, 1′-H), 5.08 (1H, dd, J3.5, 6.4, 2′-H), 4.94 (1H, d, J 6.4, 3′-H), 4.18 (2H, s, 1″-H),3.82-3.70 (2H, m, 5′-H), 2.45 (3H, s, Ts), 1.46, 1.29 (6H, s, CH₃).δ_(C) (CDCl₃) 163.6 (C-4), 150.4 (C-2), 145.2 (C-6), 142.9, 132.5,129.9, 128.0 (Ts), 114.7 (OCO), 102.6 (C5), 94.9, 87.6, 83.9, 81.5(C-4′, C-1′, C-3′, C-2′), 68.7, 63.5 (C-1″, C-5′), 26.4, 24.7 (CH₃),21.7 (Ts). FAB-MS m/z 469 [M+H]⁺.

Example 73

1-(4-C-(p-Toluenesulphonyloxymethyl-β-D-ribofuranosyl)uridine (63).Nucleoside 62 (1.33 g, 2.83 mmol) was dissolved in 80% acetic acid (25cm³) and stirred at 80° C. for 3 h whereupon the solvent was removedunder reduced pressure. The residue was coevaporated with ethanol (10cm³) and purified by silica gel column chromatography using a gradientof 0-2% methanol in dichloromethane (v/v) as eluent to give nucleoside63 as a white solid material (391 mg, 33%). δ_(H) (CD₃OD) 7.81 (1H, d, J8.1, 6-H), 7.77 (1H, d, J 8.4, Ts), 7.40 (2H, d, J 8.6, Ts), 5.74 (1H,d, J 6.6, 1′-H), 5.69 (1H, d, J 8.1, 5-H), 4.17-4.33 (2H, m, 2′-H,3′-H), 3.67-3.62 (2H, m, 1″-H), 3.26-3.20 (2H, m, 5′-H), 2.43 (3H, s,Ts). δ_(C) (CD₃OD) 166.0 (C-4), 153.0 (C-2), 146.5 (C-6), 142.5, 130.9,129.15 (Ts), 103.1 (C-5), 89.0, 87.2 (C-1′, C-4′), 75.1, 72.7, 71.3,63.8 (C-1″, C-3′, C-2′, C-5′), 21.6 (Ts).

Example 74

(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(44). To a stirred solution of nucleoside 63 (64 mg, 0.14 mmol) inanhydrous DMF (2 cm³) was slowly added sodium hydride (8.4 mg, 21 mmol,60% suspension in mineral oil, w/w) in anhydrous DMF (2 cm³) at 0° C.The reaction mixture was then heated to 120° C. for 6 h. After quenchingthe reaction with water (2 cm³), the solvents were removed under reducedpressure and the residue was purified by silica gel columnchromatography using a gradient of 5-7% methanol in dichloromethane(v/v) as eluent to give nucleoside 44 as a white solid material (9 mg,29%). NMR data were in agreement with those reported earlier forcompound 44.

Example 75

(1S,3R,4R,7S)-7-Acetoxy-1-acetoxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(64). To a stirred solution of nucleoside 37 (209.8 mg, 0.78 mmol) inanhydrous pyridine (2.5 cm³) was added acetic anhydride (0.3 cm³, 3.23mmol) and a catalytic amount of DMAP (5 mg). After stiffing for 2 h,ethanol was added (4 cm³) and the mixture was evaporated under reducedpressure. The residue was redissolved in dichloromethane and washed witha saturated aqueous solution of sodium hydrogencarbonate (7 cm³). Theorganic phase was dried (Na₂SO₄), and evaporated under reduced pressure.The residue was purified by silica gel column chromatography usingdichloromethane/methanol (97:3, v/v) as eluent affording 64 as a whitesolid material (249 mg, 90%). δ_(C) (CDCl₃) 169.59, 163.20, 149.50,133.55, 110.64, 87.05, 85.38, 77.84, 71.70, 71.02, 58.60, 20.62, 20.53,12.78. FAB-MS m/z 355 [M+H]⁺.

Example 76

(1S,3R,4R,7S)-1-Hydroxymethyl-3-(5-methyl-4-N-benzoylcytosine-1-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane(65). To a solution of nucleoside 64 (232.7 mg, 0.66 mmol) in anhydrousacetonitrile (3 cm³) was added a solution of 1,2,4-triazole (420 mg, 6.1mmol) and POCl₃ (0.12 cm³, 1.3 mmol) in anhydrous acetonitrile (5 cm³).The reaction mixture was cooled to 0° C. and anhydrous triethylamine(0.83 cm³) was added, whereupon the mixture was kept for 90 min at roomtemperature. Triethylamine (0.54 cm³) and water (0.14 cm³) was added,and the reaction mixture was stirred for 10 min and evaporated underreduced pressure. The residue was dissolved in EtOAc and washed with asaturated aqueous solution of sodium hydrogencarbonate (2×9 cm³) andwater (9 cm³). The aqueous phase was extracted with dichloromethane(3×20 cm³). The combined organic phase was evaporated under reducedpressure and the residue was redissolved in dioxane (4 cm³), whereupon32% aqueous ammonia (0.7 cm³) was added. After stiffing for 3 h, thereaction mixture was evaporated under reduced pressure and coevaporatedwith anhydrous pyridine. The residue was dissolved in anhydrous pyridine(3.6 cm³) and benzoyl chloride (0.42 cm³, 3.6 mmol) was added. Afterstirring for 2 h, the reaction was quenched with water (1 cm³) and thereaction mixture was evaporated under reduced pressure. The residue wasthen redissolved in EtOAc and washed with water (3×7 cm³). The organicphase was evaporated to dryness under reduced pressure, and the residuewas dissolved in pyridine/methanol/water (13:6:1, v/v/v, 14 cm³) at 0°C., and a 2M solution of NaOH in pyridine/methanol/water (13:6:1, v/v/v,7 cm³) was added. After stiffing for 20 min, the reaction mixture wasneutralised using a 2M solution of HCl in dioxane, and the reactionmixture was evaporated under reduced pressure. The residue was purifiedby silica column chromatography using dichloromethane/methanol (95:5,v/v) as eluent to give nucleoside 65 as a yellow foam (94.6 mg, 38%).δ_(H) (CD₃OD) 8.21 (1H, br, s), 8.02 (1H, m), 7.84-7.9 (1H, m),7.41-7.58 (4H, m), 5.61 (1H, s), 4.36 (1H, s), 4.10 (1H, s), 3.98 (1H,d, J 8.0), 3.94 (2H, s), 3.78 (1H, d, J 7.9), 2.11 (3H, d, J1.0). δ_(C)(CD₃OD, selected signals) 133.66, 132.90, 130.63, 129.50, 129.28,128.65, 90.71, 88.86, 80.57, 72.47, 70.22, 57.53, 14.01. FAB-MS m/z 374[M+H]⁺.

Example 77

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-3-(5-methyl-4-N-benzoyl-cytosine-1-yl)-7-O-(2-cyanoethoxy(diisopropylamino)phosphinoxy)-2,5-dioxabicyclo[2.2.1]heptane (66). To a stirred solutionof nucleoside 65 (82 mg, 0.22 mmol) in anhydrous pyridine (1.5 cm³) wasadded 4,4′-dimethoxytrityl chloride (80 mg, 0.24 mmol) and stirring wascontinued at room temperature for 12 h. Additional 4,4′-dimethoxytritylchloride (80 mg, 0.24 mmol) was added, and stiffing was continued foranother 12 h. Methanol (0.5 cm³) was added and the reaction mixture wasevaporated under reduced pressure. The residue was subjected to silicagel column chromatography using dichloromethane/methanol/pyridine(98.5:1.0:0.5, v/v/v). The resulting intermediate (FAB-MS m/z 676) (50.2mg) was coevaporated with anhydrous acetonitrile and dissolved inanhydrous dichloromethane (0.62 cm³). N,N-Diisopropylethylamine wasadded (0.1 cm³) followed by addition of 2-cyanoethylN,N-diisopropylphosphoramidochloridite (0.3 cm³, 0.11 mmol). Afterstiffing for 3 h at room temperature, water (1 cm³) was added and theresulting mixture was diluted with ethylacetate (10 cm³), washed withsaturated aqueous solutions of sodium hydrogencarbonate (3×6 cm³) andbrine (3×6 cm³). The organic phase was dried (Na₂SO₄) and evaporatedunder reduced pressure. The residue was purified by silica gel columnHPLC. Precipitation as described for compound 53 afforded compound 66 asa white solid material (29.5 mg, 0.03 mmol, 14%). δ_(P) (CH₃CN) 148.46,147.49.

Example 78

9-(4-(Hydroxymethyl)-2,3-O-isopropylidene-β-D-ribofuranosyl)-6-N-benzoyl-adenine(67). A mixture of oxalyl chloride (0.93 mL, 10.75 mmol) anddichloromethane (25 mL) was cooled to −70° C. Dimethyl sulfoxide (DMSO,1.7 mL, 22 mmol) was added drop-wise under intensive stirring. Themixture was stirred for 10 min at −70° C. and a solution of9-(2,3-O-isopropylidene-β-D-ribofuranosyl)-6-N-benzoyladenine (3.4 g,8.27 mmol) in dimethylsulfoxide/dichloromethane (1:9 v/v, 20 mL) wasadded during 5 min. The mixture was stirred at −60° C. for 30 min.Triethylamine (7 mL, 50.3 mmol) was added and the mixture was allowed towarm to room temperature. The solution was diluted by dichloromethane(50 mL) and washed by water (3×100 mL). Water fractions wereadditionally washed by 100 mL of dichloromethane. The organic phase wasconcentrated to an oily mass, co-evaporated with dioxane (50 mL) andre-dissolved in 30 mL of dioxane. 37% aq. formaldehyde (2.95 mL, 33.4mmol) and 2M aq. NaOH (8.26 mL) were added; the mixture was stirred atroom temperature for 10 min and cooled to 0° C. Sodium borohydride (640mg, 16.9 mmol) was added and the reaction mixture was allowed to warm toroom temperature during 15 min. The reaction was quenched by addition ofacetic acid (5 mL) and to the mixture was added dichloromethane and asaturated aqueous solution of sodium hydrogen carbonate (100 mL each).The organic phase was washed with water (100 mL), concentrated in vacuoand the product was isolated by column (2.5×25 cm) silica gelchromatography by the use of 2-3.2% of methanol in dichloromethane (v/v)as eluent. Yield 1.85 g (50.7%) of compound 67 as a white solidmaterial. δ_(H) (CDCl₃) 8.72 (1H, s), 8.14 (1H, s), 8.03-8.00 (2H, m),7.60-7.57 (1H, m), 7.56-7.46 (2H, m), 6.00 (1H, d, J4.9), 5.35 (1H, dd,J′5.8, J″5.0), 5.13 (1H, d, J5.8), 3.87-3.78 (4H, m), 1.65 (3H, s), 1.38(3H, s). δ_(C) (CDCl₃) 164.8, 152.2, 150.4, 150.2, 142.6, 133.3, 132.9,128.8, 128.0, 124.1, 114.7, 93.3, 90.2, 83.8, 82.5, 65.3, 62.9, 27.3,25.1. FAB-MS: m/z 442 [M+H]⁺, 464 [M+Na]⁺.

Alternative synthesis of 67. To a solution of2′,3′-O-isopropylideneadenosine (15 g) in anhydrous pyridine (250 mL)was added trimethylsilyl chloride (15.5 mL). The reaction mixture wasstirred at room temperature for 20 min and cooled to 0° C. Benzoylchloride (17 mL) was added drop-wise and the mixture was kept at roomtemperature temperature for 2-3 h. Water (50 mL) and 25% aq. ammoniumhydroxide (100 mL) was added and stiffing was continued for 3 h. Thenthe mixture was concentrated under reduced pressure, co-evaporated withtoluene (2×200 mL) and dichloromethane (DCM) and saturated sodiumhydrogencarbonate was added. The organic phase was evaporated to drynessto give a yellow solid. Recrystallisation from ethanol resulted in 12.5g (ca 80%) as a white solid intermediate material. Oxalyl chloride (4.68mL) in dry DCM (120 mL) was cooled to −70° C. DMSO (8.5 mL) was addedduring intensive stiffing. Later (10 min) a solution of the intermediatefor which the synthesis is described above (17 g) in 10% DMSO/DCM (100mL) added dropwise (20 min). The temperature was allowed to increase to−50° C. over a period of 30 min after which the reaction was quenchedwith triethylamine (35 mL). To the mixture was added DCM (200 ml) whichwas washed with water (3×200 mL). The intermediate was concentrated invacuo, co-evaporated with dioxane, and redissolved in dioxane (120 mL).Formaldehyde (37%) and 2 M aq. sodium hydroxide (40 mL) was added andthe reaction mixture was stirred for 1 h. The mixture was neutralisedwith acetic acid (6 mL) and DCM (400 ml) and saturated sodiumhydrogencarbonate (400 mL) were added. The organic phase wasconcentrated. The product 67 was purified by column chromatography(silica gel, 1.5-5.0% methanol/DCM). Yield 8.5 g (46%) of 67. Data wereas stated earlier in this example.

Example 79

9-(2,3-O-Isopropylidene-4-(p-toluenesulfonyloxymethyl)-β-D-ribofuranosyl)-6-N-benzoyladenine(68) and9-(4-hydroxymethyl-2,3-O-isopropylidene-5-O-(p-toluenesulfonyl)-β-D-ribofuranosyl)-6-N-benzoyladenine.A mixture of compound 67 (1.95 g, 4.42 mmol) and p-toluenesulfonylchloride (1.26 g, 6.63 mmol) was dissolved in 10 mL of anhydrouspyridine at 0° C. The reaction mixture was stirred for 4 h and thendiluted by dichloromethane (100 mL), washed with water (2×100 mL) andconcentrated under reduced pressure. The purification of the mixture bysilica gel column (2.5×20 cm) chromatography in a gradient (1-4%) ofmethanol in dichloromethane allowed isolation of starting material 67(360 mg, 18.5%) and two structural isomers, namely 68 (less polarisomer; 971 mg, 36.7%) and9-(4-hydroxymethyl-2,3-O-isopropylidene-5-O-(4′-toluenesulfonyl)-β-D-ribofuranosyl)-N⁶-benzoyladenine(more polar isomer; 352 mg, 13.1%) as white solid materials. 68: δ_(H)(CDCl₃) 8.69 (1H, s), 8.11 (1H, s), 8.00 (2H, m), 7.79 (2H, m),7.58-7.55 (1H, m), 7.50-7.46 (2H, m), 7.34-7.32 (2H, m), 5.88 (1H, d,J4.9), 5.35 (1H, dd, J′5.8, J″5.0), 5.13 (1H, d, J5.8), 3.87-3.78 (4H,m), 1.65 (3H, s), 1.38 (3H, s). δ_(C) (CDCl₃) 164.7, 152.0, 150.2,150.1, 144.9, 142.5, 133.2, 132.7, 132.3, 129.6, 128.6, 127.9, 127.8,123.9, 114.6, 93.1, 87.9, 83.4, 81.6, 68.3, 64.4, 27.1, 25.0, 21.5.FAB-MS: m/z 596 [M+H].⁺

Example 80

9-(4-(p-Toluenesulfonyloxymethyl)-β-D-ribofuranosyl)-6-N-benzoyladenine(69). A solution of compound 68 (940 mg, 1.58 mmol) in 10 mL of 90% aq.trifluoroacetic acid was kept for 30 min at room temperature andconcentrated in vacuo to an oily mass. After co-evaporation withmethanol (2×20 mL) and toluene (20 mL) the mixture was purified bysilica column (2×25 cm) chromatography in a gradient of methanol(2-5.0%) in dichloromethane as eluent to give compound 69 (825 mg, 94%)as white solid material. δ_(H) (methanol-d₄) 8.67 (1H, s), 8.53 (1H, s),8.05 (2H, d, J7.7), 7.75 (2H, d, J8.2), 7.63 (1H, m), 7.53 (2H, m), 7.32(2H, d, J8.0), 5.94 (1H, d, J 7.1), 4.92 (1H, dd, J′7.1, J″5.3), 4.41(1H, d, J5.1), 4.38 (1H, d, J10.2), 4.28 (1H, d, J10.2), 3.80 (1H, d,J12.0), 3.68 (1H, d, J12.0), 2.35 (3H, s). δ_(C) (methanol-d₄) 168.2,152.9, 150.8, 151.2, 146.4, 144.9, 134.7, 134.1, 134.0, 130.8, 129.7,129.4, 129.1, 125.1, 90.0, 88.4, 75.3, 73.1, 71.1, 64.2, 21.6. FAB-MS:m/z 556 [M+H].⁺

Example 81

9-(4-(p-Toluenesulfonyloxymethyl)-3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-β-D-ribofuranosyl)-6-N-benzoyladenine(70). To a solution of compound 69 (780 mg, 1.40 mmol) in anhydrouspyridine (7 mL) was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane(500 μL, 1.57 mmol) at 0° C. After stirring for 2 h at 0° C. additional1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (50 μL, 0.16 mmol) wasadded. The reaction mixture was allowed to warm to room temperature,diluted by dichloromethane (100 mL) and washed by water (2×100 mL). Theorganic phase was concentrated, and the residue was purified by the useof preperative HPLC (PrepPak cartridge, Porasil 15-20 μm 125 ∪; eluent:0-3% of methanol in dichloromethane (v/v) in 120 min; flow rate: 60ml/min). Concentration in vacuo yielded 870 mg (78%) of compound 70 as awhite solid material. δ_(H) (CDCl₃) 8.65 (1H, s), 8.03 (2H, m), 8.00(1H, s), 7.83 (2H, d, J8.4), 7.58 (1H, m), 7.49 (2H, m), 7.34 (2H, d,J8.4), 5.87 (1H, s), 5.70 (1H, d, J6.2), 4.68 (1H, d, J6.2), 4.59 (1H,d, J10.8), 4.31 (1H, d, J11.0), 3.91 (2H, s), 2.45 (3H, s), 1.03-0.84(28H, m). δ_(C) (CDCl₃) 164.9, 152.2, 150.5, 150.0, 144.7, 142.9, 133.5,132.9, 132.8, 129.7, 128.8, 128.1, 128.0, 123.6, 90.3, 85.3, 76.0, 75.0,68.7, 65.2, 21.6, 17.5, 17.4, 17.2, 17.1, 17.0, 16.9, 13.1, 13.0, 12.5,12.4. FAB-MS: m/z 798 [M+H].⁺

Example 82

9-(2-O,4-C-Methylene-3,5-O-(tetraisopropyldisiloxa-1,3-diyl)-β-D-ribofuranosyl)-6-N-benzoyladenine(71). A solution of compound 70 (540 mg, 0.68 mmol) in anhydrous THF (20mL) was cooled to 0° C. and sodium hydride (130 mg of 60% suspension inmineral oil, 3.25 mmol) was added under stirring. The reaction mixturewas stirred for 30 min and then neutralised by addition of 750 μL ofacetic acid. Dichloromethane (50 mL) was added, the mixture was washedby a saturated aqueous solution of sodium hydrogen carbonate (2×50 mL)and concentrated under reduced pressure. The residue was applied to asilica gel column (2.5×25 cm) and the product was eluted in a gradientconcentration (0.5 to 1.2%) of methanol in dichloromethane as eluent toyield compound 71 (356 mg, 84%) as a white foam. δ_(H) (CDCl₃) 8.77 (1H,s), 8.28 (1H, s), 8.03 (2H, m), 7.59 (1H, m), 7.50 (2H, m), 6.08 (1H,s), 4.86 (1H, s), 4.56 (1H, s), 4.14 (1H, d, J13.2), 4.06 (1H, d, J7.7),3.97 (1H, d, J13.2), 3.89 (1H, d, J7.7), 1.12-0.95 (28H, m). δ_(C)(CDCl₃) 164.8, 152.6, 150.5, 149.6, 140.7, 133.6, 132.7, 128.7, 127.9,123.1, 89.4, 86.5, 78.9, 71.7, 71.2, 56.7, 17.3, 17.1, 17.0, 16.9, 16.8,13.3, 13.1, 12.5, 12.2. FAB-MS: m/z 626 [M+H].⁺

Example 83

7-Hydroxy-1-hydroxymethyl-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(61B). Triethylamine tris-hydrofluoride (300 μL, 1.84 mmol) was added toa solution of compound 71 (420 mg, 0.067 mmol) in anhydrous THF (7 mL).The reaction mixture was kept at room temperature for 1 h andconcentrated to an oil which was purified by silica gel column (2×25 cm)chromatography eluting with 4-10% of methanol in dichloromethane (v/v).Yield 232 mg (92%) of compound 61B as a white solid material. NMR datawere identical with those reported earlier for 61B.

Example 84

1-(3,5-Di-O-benzyl-4-C-(p-toluenesulphonyloxymethyl)-2-O-p-toluene-sulphonyl-β-D-ribofuranosyl)thymine(72). A solution of1-(3,5-di-O-benzyl-4-C-(hydroxymethyl)-β-D-ribofuranosyl)thymine 35(1.48 g, 3.16 mmol), DMAP (1.344 g, 0.011 mol) and p-toluenesulphonylchloride (1.45 g, 7.6 mmol) in dichloromethane (20 ml) was stirred for 3h at room temperature. The reaction mixture was diluted withdichloromethane (30 ml) and washed with saturated aqueous solutions ofsodium hydrogen carbonate (3×20 ml) and sodium chloride (2×25 ml). Theorganic phase was dried (Na₂SO₄), filtered and evaporated under reducedpressure. The residue was subjected to silica gel column chromatographyusing methanol:dichloromethane (1:99, v/v) as eluent to give nucleoside72 (1.95 g, 80%) as a white solid material. FAB-MS m/e 776. δ_(C)(CDCl₃) 162.9, 149.8 (C-2, C-4), 145.8, 145.2 (2×Ts), 136.9, 136.8(2×Bn), 134.3 (C-6), 132.1, 132.0, 130.0, 129.9, 129.0 128.9, 128.4,128.3, 128.2, 128.0, 127.7 (2×Ts, 2×Bn), 111.2 (C-5), 85.3, 84.0 (C-1′,C-4′), 78.9, 78.3, 75.2, 74.3, 72.7, 69.1 (C-2′, C-3′, C-5′, C-1″,2×Bn), 21.7 (2×CH₃), 11.9 (CH₃). Anal. Calcd. for C₃₉H₄₀N₂S₂O₁₁: C,60.30; H, 5.19; N, 3.61. Found: C, 59.95; H, 5.11, N, 3.81.

Example 85

1-(2-Benzylamino-2-deoxy-3,5-di-O-benzyl-2-N,4-C-methylene-β-D-ribofuranosyl)thymine(73). A solution of 72 (8.6 g, 11.1 mol) in benzyl amine (10 ml) wasstirred at 130° C. for 36 h. The reaction mixture was directly subjectedto silica gel column chromatography using methanol:dichloromethane(1:99, v/v) as eluent to give nucleoside 73 (1.79 g, 30%) as a whitesolid material. FAB-MS m/e 540. δ_(C) (CDCl₃) 163.9, 149.8 (C-2, C-4),139.2, 137.6, 137.3 (3×Bn), 135.6 (C-6), 128.5, 128.4, 128.3, 128.2,128.0, 127.7, 127.0 (3×Bn), 109.6 (C-5), 88.2, 86.3 (C-1′, C-4′), 76.7,73.8, 72.0, 66.0, 63.8, 57.9, 57.8 (C-2′, C-3′, C-5′, C-1″, 3×Bn), 12.2(CH₃). Anal. Calcd. for C₃₂H₃₃N₃O₅×0.5H₂O: C, 70.06; H, 6.25; N, 7.66.Found: C, 70.00; H, 6.06; N, 7.50.

Example 86

1-(2-Amino-2-deoxy-2-N,4-C-methylene-β-D-ribofuranosyl)thymine (74). Toa solution of nucleoside 73 (1.62 g, 0.003 mol) in ethanol (150 ml) wasadded 20% palladium hydroxide on carbon (3 g) and the suspension wasstirred for 5 days under hydrogen. The catalyst was filtered off (silicagel) and washed with methanol (20 ml). The combined filtrate wasconcentrated under reduced pressure to give a white solid material whichwas filtered off and washed with methanol:dichloromethane (1:4, v/v) togive a monobenzylated intermediate (0.82 g, 76%). FAB-MS: m/e 360(M+H)⁺. ¹³C-NMR (DMSO-d₆, 250 MHz): 163.7, 149.8 (C-2, C-4), 138.2 (Bn),134.9 (C-6), 128.2, 127.5, 127.4 (Bn), 107.8 (C-5), 87.8, 87.6 (C-1′,C-4′), 72.7, 68.9, 65.9, 61.7, 49.4 (C-2′, C-3′, C-5′, C-1″, Bn), 11.9(CH₃). Anal. Calcd. for C₁₈H₂₁N₃O₅: C, 60.16; H, 5.89; N, 11.69. Found:C, 59.86; H, 5.61; N, 11.56. A mixture of this intermediate (0.1 g, 0.29mmol), ammonium formate (0.085 g, 1.35 mmol), 10% palladium on carbon(130 mg) in anhydrous methanol (7 ml) was heated under reflux for 2 h.The catalyst was filtered off (silica gel) and washed with methanol (15ml) and the combined filtrate was concentrated to dryness under reducedpressure. The residue was subjected to silica gel column chromatographyusing methanol:dichloromethane (1:9, v/v) as eluent to give titlecompound 74 (0.053 g, 71%) as a white solid material. FAB-MS m/e 270.δ_(H) (DMSO-d₆) 11.29 (bs, 1H, NH), 7.73 (d, 1H, J1.1, 6-H), 5.31 (s,1H, 1′-H), 5.29 (br s, 1H, 3′-OH), 5.13 (m, 1H, 5′-OH), 3.81 (s, 1H,3′-H), 3.69 (m, 2H, 5′-H), 3.23 (s, 1H, 2′-H), 2.88 (d, 1H, J 9.8,1″-H_(a)), 2.55 (d, 1H, J 9.8, 1″-H_(b)), 1.77 (d, 3H, J 0.8, CH₃).δ_(C) (DMSO-d₆) 164.0, 150.1 (C-2, C-4), 135.6 (C-6), 107.8 (C-5), 89.5,87.9 (C-1′, C-4′), 68.7, 61.9, 57.1, 49.4, (C-2′, C-3′, C-5′, C-1″).Anal. Calcd. for C₁₁H₁₅N₃O₅×0.5H₂O: C, 47.48; H, 5.80; N, 15.10. Found:C, 47.54; H, 5.30; N, 14.79.

Alternative method for conversion of 73 to 74. To a solution of 73(0.045 g, 0.0834 mmol) in methanol (6 ml) was added 10% Pd on carbon(0.118 g) and—in three portions during 3 h—ammonium formate (0.145 g,0.0023 mol). The suspension was refluxed for 4.5 h. The catalyst wasfiltered off (silica gel) and washed with methanol (4×3 ml). Thecombined filtrate was concentrated and the residue was subjected tocolumn chromatography on silica gel using methanol:dichloromethane (1:9,v/v) as eluent to give nucleoside 74 (0.015 g, 67%). Spectral data werein accordance with data reported earlier in this example for 74.

Example 87

1-(2-Amino-2-deoxy-2-N,4-C-methylene-2-N-trifluoroacetyl-β-D-ribofuranosyl)thymine(74-COCF₃). To a suspension of nucleoside 74 (0.050 g, 0.186 mmol) inmethanol (2 mL) were added DMAP (0.013 mg, 0.106 mmol) and ethyltrifluoroacetate (0.029 mL, 0.242 mmol) and the mixture was stirred atroom temperature for 2.5 h. The solvent was removed under reducedpressure and the residue was subjected to column chromatography onsilica gel using methanol:dichloromethane (2.5:97.5, v/v) as eluent togive the title nucleoside 74-COCF₃ as a white solid material afterevaporation of the solvents under reduced pressure (0.055 g, 81%).FAB-MS m/z 366 [M+H]⁺. ¹³C NMR (CD₃OD, δ2.9 MHz) δ 166.5, 157.7 (q,²J_(C,F) 37.5 Hz, COCF₃), 157.6 (q, ²J_(C,F) 37.2 Hz, COCF₃), 151.8,136.8, 136.8, 117.6 (d, ¹J_(C,F) 287.5 Hz, CF₃), 117.5 (d, ¹J_(C,F)286.5 Hz, CF₃), 110.8, 110.8, 90.7, 89.3, 87.7, 87.3, 70.1, 68.6, 66.2,66.2, 64.5, 57.9, 53.3, 12.7. Anal. Calcd. for C₁₃H₁₄N₃O₆F₃: C, 42.8; H,3.9; N, 11.5. Found: C, 42.5; H, 4.0; N, 11.2.

Example 88

1-(2-Amino-2-deoxy-5-O-4,4′-dimethoxytrityl-2-N,4-C-methylene-2-N-trifluoroacetyl-β-D-ribofuranosyl)thymine(74-DMT). To a solution of nucleoside 74-COCF₃ (0.030 g, 0.082 mmol) inanhydrous pyridine (0.6 mL) at 0° C. was dropwise (during 20 min) added4,4′-dimethoxytrityl chloride (0.054 g, 0.159 mmol) dissolved inanhydrous pyridine:dichloromethane (0.6 mL, 1:1, v/v) and the mixturewas stirred for 10 h at room temperature. A mixture of ice and water wasadded (5 mL) and the resulting mixture was extracted withdichloromethane (3×5 mL). The combined organic phase was washed with asaturated aqueous solution of sodium hydrogencarbonate (3×2 mL), dried(Na₂SO₄) and filtered. The filtrate was evaporated to dryness underreduced pressure and the residue was subjected to column chromatographyon silica gel using methanol:dichloromethane:pyridine (1.5:98.0:0.5,v/v/v) as eluent to give nucleoside 74-DMT as a white solid materialafter evaporation of the solvents under reduced pressure (0.051 g, 93%).FAB-MS m/z 667 [M]⁺, 668 [M+H]⁺.FAB-HRMS calcd. for C₃₄H₃₂N₃O₈F₃ ⁺:667.2142. Found: 667.2146. ¹³C NMR(C₅D₅N, 100.6 MHz) δ 165.1, 165.0,159.5, 159.5, 151.4, 145.7, 136.3, 136.1, 134.8, 134.6, 130.9, 130.9,130.9, 128.9, 128.9, 128.7, 128.7, 128.4, 127.7, 123.2, 114.1, 114.1,114.0, 110.4, 89.4, 87.9, 87.5, 87.4, 87.2, 70.8, 69.0, 66.0, 64.4,60.5, 60.2, 55.5, 53.6, 53.4, 49.9, 13.2, 13.1.

Example 89

1-(2-Amino-3-O-(2-cyanoethoxy(diisopropylamino)phosphino-2-deoxy)-5-O-4,4′-dimethoxytrityl-2-N,4-C-methylene-2-N-trifluoroacetyl-β-D-ribofuranosyl)-thymine(74A). To a solution of nucleoside 74-DMT (0.121 g, 0.181 mmol) inanhydrous dichloromethane (2 mL) were added N,N-diisopropylethylamine(0.093 mL, 0.54 mmol) and 2-cyanoethylN,N-diisopropylphosphoramidochloridite (0.057 mL, 0.26 mmol) at 0° C.and the mixture was stirred for 10 h at room temperature. The mixturewas diluted with dichloromethane (20 mL), extracted with a saturatedaqueous solution of sodium hydrogencarbonate (3×10 mL), dried (Na₂SO₄)and filtered. The filtrate was evaporated to dryness under reducedpressure and the residue was subjected to column chromatography onsilica gel using methanol:dichloromethane:pyridine (1.5:98.0:0.5, v/v/v)as eluent to give crude product (0.107 g) after evaporation of thesolvents under reduced pressure. The residue was dissolved in anhydrousdichloromethane (1 mL) and by dropwise addition to vigorously stirredpetroleum ether (60-80° C., 30 mL) at −30° C., nucleotide 74Aprecipitated to give a white solid material after filtration (0.090 g,57%). FAB-MS m/z 868 [M+H]⁺, 890 [M+Na]⁺. ³¹P NMR (CD₃CN, 121.5 MHz) δ150.4, 150.2, 148.8, 149.1.

Example 90

1-(2-Amino-2-N,4-C-methylene-3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-β-D-ribofuranosyl)thymine(74B). To a solution of nucleoside 74 (0.20 g, 0.74 mmol) in anhydrouspyridine (3 mL) at −15° C. was dropwise (during 3 h) added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.305 mL, 0.0011 mol) andthe mixture was stirred for 10 h at room temperature. MeOH (3 mL) wasadded and the mixture was evaporated to dryness under reduced pressure.The residue was subjected to column chromatography on silica gel usingmethanol:dichloromethane (1:99, v/v) to give nucleoside 74B as a whitesolid material after evaporation of the solvents under reduced pressure(0.370 mg, 97%). FAB-MS m/z 512 [M+H]⁺. ¹H NMR((CD₃)₂SO, 400 MHz) δ11.37 (bs, 1H), 7.48 (s, 1H), 5.32 (s, 1H), 4.06 (d, 1H, J 13.5 Hz),4.00 (s, 1H), 3.84 (d, 1H, J 13.5 Hz), 3.41 (s, 1H), 2.92 (d, 1H, J 10.2Hz), 2.64 (d, 1H, J 10.2 Hz), 1.74 (s, 3H), 1.10-0.92 (m, 28H). ¹³CNMR((CD)₃SO₂, 62.9 MHz) δ 163.8, 149.8, 134.1, 107.9, 89.5, 87.9, 70.1,61.1, 57.9, 49.3, 17.2, 17.2, 17.0, 16.9, 16.8, 16.7, 12.6, 12.2, 11.7.Anal. Calcd. for C₂₃H₄₁N₃O₆Si₂: C, 54.0; H, 8.1; N, 8.2. Found: C, 54.0;H, 8.3; N, 7.8.

Example 91

1-(2-Deoxy-2-methylamino-2-N,4-C-methylene-3,5-O-(tetraisopropyldisil-oxane-1,3-diyl)-β-D-ribofuranosyl)thymine(74C). To a solution of nucleoside 74B (0.33 g, 0.64 mmol) in anhydrousTHF:dichloromethane (4:1, v/v) at −10° C. was dropwise (during 30 min)added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.125 mL, 0.836 mmol) andmethyl iodide (0.05 mL, 0.79 mmol) and the mixture was stirred for 48 hat 10° C. Additional DBU (0.05 mL, 0.33 mmol) and methyl iodide (0.020mL, 0.32 mmol) was dropwise (during 15 min) added to the reactionmixture and stirring at 10° C. was continued for 24 h. The mixture wasevaporated to dryness under reduced pressure and the residue wassubjected to column chromatography on silica gel usingmethanol:dichloromethane (1:99, v/v) as eluent to give nucleoside 74C asa white solid material after evaporation of the solvents (0.25 g, 74%).FAB-MS m/z 526 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz) δ 8.19 (bs, 1H), 7.65 (d,1H, J 1.3 Hz), 5.59 (s, 1H), 4.11 (s, 1H), 4.05 (d, 1H, J 13.2 Hz), 3.87(d, 1H, J 13.2 Hz), 3.44 (s, 1H), 2.98 (d, 1H, J 9.5 Hz), 2.71 (d, 1H, J9.5 Hz), 2.72 (s, 3H), 1.91 (d, 1H, J 1.1 Hz), 1.12-0.96 (m, 28H). ¹³CNMR (CDCl₃, 62.9 MHz) δ 163.7, 149.6, 135.2, 109.7, 90.9, 85.7, 71.4,67.3, 58.6, 58.2, 41.2, 17.5, 17.4, 17.3, 17.2, 17.1, 16.9, 13.3, 13.1,13.0, 12.6, 12.1. Anal. Calcd. for C₂₄H₄₄N₃O₆Si₂,0.25H₂O: C, 54.4; H,8.3; N, 7.9. Found: C, 54.4; H, 8.1; N, 7.7.

Example 92

1-(2-Deoxy-2-methylamino-2-N,4-C-methylene-β-D-ribofuranosyl)thymine(74D). To a solution of nucleoside 74C (0.40 g, 0.76 mmol) in THF atroom temperature was added a solution of tetrabutylammonium fluoride inTHF (1.0 M, 2.2 mL, 2.2 mmol) and the reaction mixture was stirred for20 min whereupon pyridine:water:methanol (6 mL, 3:1:1, v/v/v) was added.The mixture was added to Dowex 50×200 resin (2.2 g, H⁺ (pyridinium)form, 100-200 mesh) suspended in pyridine:water:methanol (6 mL, 3:1:1,v/v/v) and the resulting mixture was stirred for 20 min. Afterfiltration, the residue was washed with pyridine:water:methanol (3×3 mL,3:1:1, v/v/v) and the combined filtrate was evaporated to dryness underreduced pressure to give an oily residue after coevaporation withmethanol (2×5 mL). Column chromatography on silica gel usingmethanol:dichloromethane (1:49, v/v) as eluent gave nucleoside 74D as awhite solid material after evaporation of the solvents under reducedpressure (0.17 g, 79%). FAB-MS m/z 284 [M+H]⁺. FAB-HRMS calcd. forC₁₂H₁₈N₃O₅ ⁺: 284.12465. Found: 284.12402. ¹H NMR((CD₃)₂SO, 400 MHz) δ11.3 (bs, 1H, NH), 7.70 (d, 1H, J 1.1 Hz, 6-H), 5.50 (s, 1H, 1′-H), 5.26(d, 1H, J 4.9 Hz, 3′-OH), 5.12 (t, 1H, J 5.7 Hz, 5′-OH), 3.87 (d, 1H, J4.8 Hz, 3′-H), 3.67 (d, 2H, J 5.5 Hz, 5′-H), 3.12 (s, 1H, 2′-H), 2.87(d, 1H, J 9.3 Hz, 5″—H_(a)), 2.56 (s, 3H, NCH₃), 2.52-2.49 (1H, m,5″-H_(b)), 1.77 (s, 3H, CH₃). ¹H NMR (CD₃OD, 400 MHz) δ 7.80 (d, 1H, J1.3 Hz, 6-H), 5.71 (s, 1H, 1′-H), 4.07 (s, 1H, 3′-H), 3.83 (s, 2H,5′-H), 3.36 (s, 1H, 2′-H), 3.08 (d, 1H, J 9.9 Hz, 5″-H_(a)), 2.68 (s,3H, NCH₃), 2.57 (d, 1H, J 9.9 Hz, 5″-H_(b)), 1.88 (d, 3H, J 1.1 Hz,CH₃). ¹³C NMR (CD₃OD, δ2.9 MHz) δ 166.6, 151.9, 137.4, 110.4, 91.3,85.2, 71.4, 69.1, 59.4, 58.7, 40.2, 12.2.

Example 93

1-(2-Deoxy-5-O-4,4′-dimethoxytrityl-2-methylamino-2-N,4-C-methylene-β-D-ribofuranosyl)thymine(74E). To a solution of nucleoside 74D (0.135 g, 0.477 mmol) inanhydrous pyridine (1.5 mL) at 0° C. was dropwise (during 20 min) addeda solution of 4,4′-dimethoxytrityl chloride (0.238 g, 0.702 mmol) inanhydrous pyridine:dichloromethane (1.0 mL, 1:1, v/v) and the resultingmixture was stirred for 10 h at RT. A mixture of ice and water was added(5 mL) and the mixture was extracted with dichloromethane (3×10 mL). Thecombined organic phase was washed with a saturated aqueous solution ofsodium hydrogencarbonate (3×5 mL), dried (Na₂SO₄) and filtered. Thefiltrate was evaporated to dryness under reduced pressure and theresidue was subjected to column chromatography on silica gel usingmethanol:dichloromethane:pyridine (1:98:1, v/v/v) as eluent to givenucleoside 74E as a white solid material after evaporation of thesolvents under reduced pressure (0.20 g, 72%). FAB-MS m/z 586 [M+H]⁺. ¹HNMR(C₅D₅N, 400 MHz) δ 13.2 (bs, 1H), 7.98 (d, 1H, J 1.3 Hz), 7.98-7.00(m, 13H), 6.12 (s, 1H), 4.78 (d, 1H, J 3.7 Hz), 3.88-3.79 (m, 4H), 3.71(s, 3H), 3.71 (s, 3H), 3.29 (d, 1H, J 9.3 Hz), 2.84 (d, 1H, J 9.3 Hz),2.81 (s, 3H), 1.85 (d, 3H, J 0.9 Hz). ¹³C NMR(C₅D₅N, 62.9 MHz) δ 165.1,159.2, 151.4, 145.9, 136.5, 136.4, 130.8, 130.7, 128.7, 128.4, 127.4,113.8, 109.6, 89.8, 86.8, 85.1, 72.0, 68.7, 60.9, 59.4, 55.2, 40.1,13.1. Anal. Calcd. for C₃₃H₃₅N₃O₇,0.25H₂O: C, 67.2; H, 6.1; N, 7.1.Found: C, 67.2; H, 6.2; N, 6.9.

Example 94

1-(3-O-(2-Cyanoethoxy(diisopropylamino)posphino)-5-O-4,4′-dimethoxytrityl-2-methylmino-2-N,4-C-methylene-2-deoxy-β-D-ribofuranosyl)thymine(74F). To a solution of nucleoside 74E (0.130 g, 0.222 mmol) inanhydrous dichloromethane (2 mL) at 0° C. were addedN,N-diisopropylethylamine (0.088 mL, 0.514 mmol) and 2-cyanoethylN,N-diisopropylphosphoramidochloridite (0.065 mL, 0.291 mmol) and themixture was stirred for 10 h at room temperature. Dichloromethane (30mL) was added and the mixture was extracted with a saturated aqueoussolution of sodium hydrogencarbonate (3×10 mL), dried (Na₂SO₄) andfiltered. The filtrate was evaporated to dryness under reduced pressureand the residue was subjected to column chromatography on silica gelusing methanol:dichloromethane:pyridine (0.5:98.5:1.0, v/v/v) as eluentto give crude product (0.120 g) after evaporation of the solvents underreduced pressure. The residue was dissolved in anhydrous dichloromethane(1 mL) and by dropwise addition to vigorously stirred petroleum ether(60-80° C., 30 mL) at −30° C., nucleotide 74F precipitated to give awhite solid material after filtration (0.090 g, 52%). ³¹P NMR (CD₃CN,121.5 MHz) δ 147.7.

Example 95

1-(3,5-Di-O-benzyl-4-C-(p-toluenesulphonyloxymethyl)-2-O-p-toluene-sulphonyl-β-D-ribofuranosyl)uracil(75). To a stirred solution of1-(3,5-di-O-benzyl-4-C-hydroxymethyl-(3-D-ribofuranosyl)uracil 41 (3.55g, 7.81 mmol) in dichloromethane (50 cm³) were added DMAP (3.82 g) andp-toluenesulphonyl chloride (4.47 g, 23.5 mmol) at room temperature.Stirring was continued for 2 h, and dichloromethane (100 cm³) was added.The reaction mixture was washed with a saturated aqueous solution ofsodium hydrogen carbonate (2×75 cm³) and dried (Na₂SO₄). The organicphase was evaporated under reduced pressure and the residue was purifiedby silica gel column chromatography using dichloromethane/methanol(99.5:0.5, v/v) as eluent to give nucleoside 75 (4.65 g, 78%) as a whitesolid material. δ_(H) (CDCl₃) 8.49 (1H, br s, NH), 7.67 (1H, d, J 8.3,6-H), 7.51-7.03 (18H, m, Bn, Ts), 6.0 (1H, d, J 7.6, 1′-H), 5.05 (1H, m,2′-H), 4.91 (2H, m, 5-H, Bn), 4.56 (2H, m, Bn), 4.42 (1H, d, J 10.4,Bn), 4.31 (1H, d, J 4.9, 3′-H), 4.05 (2H, m, 1″-H), 3.75-3.64 (2H, m,5′-H), 2.41 (3H, s, CH₃), 2.34 (3H, s, CH₃). δ_(C) (CDCl₃) 162.2 (C-4),149.5 (C-2), 146.0, 145.3 (Ts), 139.0 (C-6), 136.7, 131.9, 130.0, 129.9,128.9, 128.7, 128.5, 128.4, 128.3, 128.2, 128.0, 127.6 (Bn, Ts) 102.7(C-5), 85.5 (1′-C), 84.4 (4′-C), 79.2, 78.3, 75.1, 74.3, 72.4, 69.1 (Bn,3′-C, 2′-C, 5′-C, 1″-C), 21.7, 21.6 (Ts). FAB-MS m/z 763. Found: C,61.2; H, 4.4; N, 3.3; C₃₈H₃₈N₂O₁₁S₂ requires C, 59.8; H, 5.0; N, 3.6.

Example 96

1-(2-Deoxy-3,5-di-O-benzyl-2-S,4-C-methylene-2-mercapto-(3-D-ribofura-nosyl)thymine(76). To a stirred solution of nucleoside 75 (3.70 g, 4.86 mmol) in DMF(40 cm³) was added potassium thioacetate (0.83 g, 7.28 mmol). Themixture was stirred and heated at 110° C. for 80 h. After evaporationunder reduced pressure, H₂O (100 cm³) was added. Extraction wasperformed with dichloromethane (4×50 cm³) and the combined organic phasewas dried (Na₂SO₄), filtered and evaporated under reduced pressure. Theresidue was purified by silica gel chromatography usingdichloromethane/methanol (99.6:0.4, v/v) as eluent to give nucleoside 76(1.65 g, 75%) as a white solid material. δ_(H) (CDCl₃) 9.08 (1H, br s,NH), 7.98 (1H, d, J 8.1, 6-H), 7.39-7.20 (10H, m, Bn), 5.85 (1H, s,1′-H), 5.26 (1H, d, J 8.1, 5-H), 4.61 (1H, d J 11.4, 5′-H), 4.56 (2H, s,Bn), 4.45 (1H, d, J 11.4, Bn), 4.14 (1H, d, J 1.7, 3′-H), 3.82 (2H, m,Bn), 3.72 (1H, d, J 1.9, 2′-H), 3.02 (1H, d, J 9.9, 1″-H_(a)), 2.78 (1H,d, J 9.9, 1″-H_(b)). δ_(C) (CDCl₃) 163.4 (C-4), 150.0 (C-2), 139.9(C-6), 137.2, 136.8, 128.6, 128.5, 128.2, 127.9, 127.7 (Bn), 100.8(C-5), 90.8, 88.8 (C-1′, C-4′), 76.5, 73.8, 72.0, 70.0 (2×Bn, C-3′,C-5′), 49.52 (C-2′), 35.63 (C-1″). FAB-MS m/z 453. Found: C, 63.4; H,5.1; N, 5.9; C₂₄H₂₄N₂O₅S requires C, 63.7; H, 5.3; N, 6.1.

Example 97

1-(2-O-p-Toluenesulfonyl-4-C-(p-toluenesulfonyloxymethyl)-β-D-ribofura-nosyl)uracil(76A). To a solution of compound 75 (0.80 g, 1.0 mmol) in absoluteethanol (2 cm³) was added 20% palladium hydroxide over carbon (0.80 g)and the mixture was degassed several times with hydrogen and stirringwas continued under hydrogen for 48 h. The catalyst was filtered off andthe filtrate was evaporated under reduced pressure. The residue waspurified by silica gel column chromatography usingdichloromethane/methanol (99:1, v/v) as eluent to give nucleoside 76A(0.30 g, 49%) as a white solid material. δ_(H) (CD₃OD) 7.67 (4H, m),7.45 (1H, d, J 8.2 Hz), 7.34 (4H, m), 5.86 (1H, d, J 8.0 Hz), 5.40 (1H,d, J 8.1 Hz), 4.95 (1H, m), 4.35 (1H, d, J 5.0 Hz), 4.17 (2H, m), 3.61(2H, s), 2.40 (6H, s). δ_(C) (CD₃OD) 165.4, 151.6, 147.5, 146.6, 141.3,134.0, 133.8, 131.4, 130.9, 129.2, 128.9, 103.7, 88.0, 85.4, 80.7, 72.4,71.0, 64.3, 21.7, 21.6. FAB-MS m/z 583 [M+H]⁺.

Example 98

1-(3,5-O-(Tetraisopropyldisiloxa-1,3-diyl)-2-O-p-toluenesulfonyl-4-C-(p-toluenesulfonyloxymethyl)-β-D-ribofuranosyl)uracil(76B). To a stirred solution of nucleoside 76A (0.27 g, 0.46 mmol) inanhydrous pyridine (4 cm³) was added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.22 cm³, 0.70 mmol).After stirring for 48 h, the mixture was cooled to 0° C. and a saturatedaqueous solution of sodium hydrogen carbonate (15 cm³) was added. Themixture was extracted with dichloromethane (3×10 cm³) and the combinedorganic phase was dried (Na₂SO₄) and filtered. The solvent wasevaporated under reduced pressure and the residue was purified by silicagel chromatography using dichloromethane/methanol (99.5:0.5, v/v) aseluent to give nucleoside 76B (0.37 g, 97%) as a white solid material.δ_(H) (CDCl₃) 8.70 (1H, br s), 7.80 (4H, m), 7.36 (4H, m), 6.98 (1H, d,J 8.1 Hz), 5.64 (1H, d, J 8.0 Hz), 5.18 (2H, m), 4.98 (1H, d, J 7.0 Hz),4.39-4.32 (2H, m), 3.92-3.76 (2H, s), 2.45 (6H, s), 1.27-0.66 (28H, m).δ_(C) (CDCl₃) 162.9, 149.3, 145.6, 144.8, 143.9, 132.9, 130.1, 129.9,128.2, 128.1, 102.2, 94.6, 84.7, 80.4, 72.8, 67.8, 64.6, 21.7, 17.3,17.2, 17.1, 16.9, 16.8, 13.1, 12.8, 12.3. FAB-MS m/z 825 [M+H]⁺.

Example 99

1-(2-Deoxy-2-mercapto-2-S,4-C-methylene-3,5-O-(tetraisopropyldisiloxa-1,3-diyl)-β-D-ribofuranosyl)uracil(76C). To a stirred solution of nucleoside 76B (0.26 g, 0.32 mmol) inDMF (5 cm³) was added potassium thioacetate (0.054 g, 0.47 mmol). Thereaction mixture was stirred at 110° C. for 20 h. After evaporation ofthe mixture under reduced pressure, H₂O (20 cm³) was added. Extractionwas performed with dichloromethane (3×10 cm³) and the combined organicphase was dried (Na₂SO₄), filtered and evaporated under reducedpressure. The residue was purified by silica gel column chromatographyusing dichloromethane/methanol (99.25:0.75, v/v) as eluent to givenucleoside 76C (0.125 g, 77%) as a white solid material. δ_(H) (CDCl₃)8.55 (1H, br s), 8.02 (1H, d, J 8.1 Hz), 5.82 (1H, s, 1′-H), 5.65 (1H,d, J 8.1 Hz), 4.37 (1H, d, J 2.1 Hz), 4.10 (1H, d, J 13.2 Hz), 3.90 (1H,d, J 13.1 Hz), 3.53 (1H, d, J 2.1 Hz), 2.92 (1H, d, J 10.1 Hz), 2.74(1H, d, J 10.0 Hz), 1.30-0.80 (28H, m). δ_(C) (CDCl₃) 163.2, 149.8,139.6, 100.9, 91.4, 90.7, 71.5, 59.8, 51.5, 34.4, 17.5, 17.3, 17.1,16.9, 15.5, 13.6, 13.3, 13.1, 12.9, 12.3. FAB-MS m/z 515 [M+H]⁺.

Example 100

1-(2-Deoxy-2-mercapto-2-S,4-C-methylene-β-D-ribofuranosyl)uracil (76D).To a stirred solution of nucleoside 76C (25 mg, 0.049 mmol) in THF (1.0cm³) was added a solution of tetrabutylammonium flouride (0.20 cm³ of a1M solution in THF, 0.20 mmol) at 0° C. After stirring the mixture at 0°C. for 1 h, H₂O (5 cm³) was added and the mixture was evaporated. Theresidue was purified by silica gel column chromatography usingdichloromethane/methanol (97:3, v/v) as eluent to give nucleoside 76D(9.0 mg, 69%) as a white solid material. δ_(H) (CD₃OD) 8.19 (1H, d, J8.1 Hz, 6-H), 5.77 (1H, s, 1′-H), 5.65 (1H, d, J 8.1 Hz, 5-H), 4.31 (1H,d, J 2.1 Hz, 3′-H), 3.86 (2H, s, 5′-H), 3.53 (1H, d, J 2.2 Hz, 2′-H),2.93 (1H, d, J 10.3 Hz, 1″-H_(a)), 2.73 (1H, d, J 10.3 Hz, 1″-H_(b)).δ_(C) (CD₃OD) 166.5, 152.0, 141.7, 101.2, 92.1, 92.0, 71.4, 59.9, 53.6,35.4. FAB-MS m/z 273 [M+H]⁺.

Example 101

1-(2-Deoxy-5-O-(4,4′-dimethoxytrityl)-2-mercapto-2-S,4-C-methylene-β-D-ribofuranosyl)uracil(76E). To a solution of 76D (0.2 g, 0.37 mmol) in anhydrous pyridine (5cm³) was added 4,4′-dimethoxytrityl chloride (0.186 g, 0.55 mmol) atroom temperature. The solution was stirred for 5 h whereupon thereaction mixture was cooled to 0° C. A saturated aqueous solution ofsodium hydrogen carbonate (30 cm³) was added and the resulting mixturewas extracted with dichloromethane (3×50 cm³). The combined organicphase was separated and dried (Na₂SO₄). The solvent was removed underreduced pressure and the residue was purified by silica gel columnchromatography with dichloromethane/methanol/pyridine (98.5:1.0:0.5 v/v)as eluent to give nucleoside 76E as a white brownish solid material(0.175 g, 83%). δ_(C) (CDCl₃) 164.5, 159.4, 151.6, 145.7, 139.9, 136.4,136.0, 135.6, 130.9, 130.8, 128.8, 128.5, 128.4, 127.5, 127.4, 122.7,113.9, 101.5, 91.7, 90.2, 87.6, 71.8, 61.9, 55.3, 53.7, 36.2, 30.6.FAB-MS m/z 574 [M]⁺, 575 [M+H]⁺ (Found: C, 65.2; H, 5.4; N, 5.0;C₃₁1^(˜)1₃₀N₂O₇S requires C, 64.8; H, 5.3; N, 4.9%).

Example 102

1-(3-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-(2-deoxy-5-O-(4,4′-dimethoxytrityl)-2-mercapto-2-S,4-C-methylene-β-D-ribofuranosyl)uracil(76F). To a solution of 76E (0.160 g, 0.28 mmol) in anhydrousdichloromethane (2 cm³) at 0° C. were added N,N-diisopropylethylamine(0.27 cm³) and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (97mg, 0.42 mmol). Stirring was continued at room temperature for 5 h. Thereaction mixture was cooled to 0° C. and a saturated aqueous solutionsof sodium hydrogen carbonate (30 cm³) was added. Extraction wasperformed using dichloromethane (3×20 cm³) and the combined organicphase was dried (Na₂SO₄) and evaporated to dryness under reducedpressure. The residue was purified by silica gel column chromatographyusing dichloromethane/methanol/pyridine (99:0.5:0:5 v/v) as eluent togive a white foam. This residue was dissolved in dichloromethane (2 cm³)and the product was precipitated from light petroleum (100 cm³, cooledto −40° C.) under vigorous stiffing. The precipitate was collected byfiltration, and was finally dried to give nucleoside 76F as a whitesolid material (95 mg, 44%). δ_(P) (CDCl₃) 148.9, 149.0.

Example 103

3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-(p-toulenesulfonyloxy-methyl)-β-D-ribofuranose(77).A solution of3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose31 (15.38 g, 38.4 mmol), anhydrous pyridine (20 cm³) and anhydrousdichloromethane (80 ml) was stirred at −5° C. p-Toulenesulphonylchloride (8.75 g, 46.0 mmol) dissolved in anhydrous dichloromethane (8cm³) was added during 15 min. The solution was stirred at roomtemperature for 17 h. The reaction was quenched with ice-cold H₂O (200cm³). Extraction was performed with dichloromethane (5×150 cm³) and thecombined organic phase was washed with saturated aqueous solutions ofsodium hydrogen carbonate (3×100 cm³) and brine (3×100 cm³), dried(Na₂SO₄), filtered and evaporated under reduced pressure. The residuewas purified by silica gel column chromatography usingdichloromethane:methanol (98.5:1.5, v/v) as eluent to give 77 as a clearoil (17.4 g, 82%). δ_(H) (CDCl₃) 7.79-7.19 (14H, m, Bn), 5.66 (1H, d, J3.6, 1-H), 4.69-4.20 (8H, m, Bn, 5-H_(a), 5-H_(b), 3-H, 2-H), 3.53 (1H,d, J 10.3, 1′-H_(a)), 3.46 (1H, d, J 10.3, 1′-H_(b)), 2.40 (3H, s, CH₃),1.29 (3H, s, CH₃), 1.26 (3H, s, CH₃). δ_(C) (CDCl₃) 144.6, 137.9, 137.3,133.0, 129.8, 128.4, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6(aromatic), 113.6 (C(CH₃)₂), 104.2, (C-1), 84.7 (C-4), 79.0, 78.7, 73.7,72.7, 70.7, 70.2, (Bn, C-2, C-3, C-5, C-1′), 26.3, 26.0 (C(CH₃)₂), 21.6(CH₃). FAB-MS m/z 555 [M+H]⁺. (Found: C, 64.8; H, 6.2; C₃₀H₃₄O₈Srequires C, 64.9; H, 6.1%).

Example 104

1,2-Di-O-acetyl-3,5-di-O-benzyl-4-C-(p-toluenesulfonyloxymethyl)-α,β-D-ribofuranose(78). A solution of furanose 77 (17.4 g, 31.4 mmol) in 80% acetic acid(250 cm³) was stirred at 60° C. for 20 h. The solvent was removed invacuo and the residue was coevaporated with toluene (3×20 cm³). Theresidue was redissolved in anhydrous pyridine (100 cm³). Aceticanhydride (14.2 cm³) was added and the solution was stirred for 15 h atroom temperature. The reaction was quenched by addition of ice-cold H₂O(200 cm³), and the mixture was extracted with dichloromethane (4×150cm³). The combined organic phase was washed with saturated aqueoussolutions of sodium hydrogen carbonate (2×125 cm³) and brine (3×150cm³), dried (Na₂SO₄), filtered and evaporated under reduced pressure.The residue was purified by silica gel column chromatography usingdichloromethane:methanol (98.5:1.5, v/v) as eluent to give 78 (α,β˜1:1)as a clear oil (13.5 g, 72%). δ_(C) (CDCl₃) 169.8, 169.6, 69.4, 168.8(C═O), 144.7, 137.7, 137.5, 132.8, 129.7, 129.6, 128.5, 128.4, 128.3,128.2, 128.0, 127.8, 127.7, 127.6 (Bn), 97.4, 94.2 (C-1), 86.4, 84.2(C-4), 78.9, 77.5, 74.5, 74.1, 73.7, 73.5, 71.8, 70.6, 70.5, 69.6, 69.5(Bn, C-2, C-3, C-1′), 21.6, 21.0, 20.8, 20.6, 20.4 (COCH₃, C(CH₃)₂).FAB-MS m/z 599 [M+H]⁺.

Alternative Procedure for the Preparation of Compound 78.

3-O-Benzyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (30B). To asolution of 1,2:5,6-Di-O-isopropylidene-α-D-allofuranose (30A) (obtainedfrom Pfanstiehl Laboratories Inc.) (40 g) in dimethylformamide at 0° C.was added sodium hydride in smaller portions. The reaction mixture wasstirred for 1 h, benzyl bromide was added drop wise over a period of 1h. The reaction mixture was stirred at room temperature for 16 h.Methanol was added to quench the reaction and dimethylformamide wasremoved under pressure. The syrup was extracted with ethyl acetate andwashed with brine. Evaporation of the ethyl acetate layer yielded asemisolid (93%). Homogeneous by TLC.

3-O-Benzyl-1,2-O-isopropylidene-α-D-glucofuranose (30C). Partialhydrolysis of 30B (50 g) was achieved in 75% acetic acid in a period of20 h. Concentration to a smaller volume and extraction with ethylacetate yielded 30C, 40 g, (90%). Homogeneous by TLC.

3-O-Benzyl-1,2-O-isopropylidene-α-D-ribo-pentodialdofuranose (30D). Asolution of 30C (40 g) in water/methanol (1:1) was slowly added withstiffing to a solution of sodium periodate in water at 0° C. Thereaction was stirred for 2 h, ethylene glycol was added and the mixturewas extracted with ethyl acetate. The dried extract was evaporated toyield 30D, 32 g, (89%). Homogeneous by TLC. In this step addition ofmethanol is essential for the completion of the reaction.

3-O-Benzyl-4-(hydroxymethyl)-1,2-O-isopropylidene-α-D-erythro-pentofuranose(30E). Aqueous 37% formaldehyde and 1N sodium hydroxide were added at 0°C. to a stirred solution of 30D (32 g) in water and tetrahydrofuran(1:1), the reaction was continued for 16 h, extracted in ethyl acetateand washed with brine. Evaporation of the organic layer afforded a syrupwhich crystallised from ether/petroleum ether as white solid, 23 g, thefiltrate was an oil which solidified as a low melting solid, 10 g, totalyield of 30E, 92%. [23 g (white solid was 99% pure by TLC), 10 g of lowmelting solid (had faster moving impurities by TLC, approximately 75%pure)]. In this step addition of tetrahydrofuran is very important forthe time and reaction completion.

3,5-Di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose(31). Benzylation of 30E (20 g) with NaH 60% and BnBr at −10° C. yieldeda mixture of two isomers. Flash column chromatography afforded 31 as themajor isomer, 14 g, (54%). Homogeneous by TLC.

3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-tosyl-α-D-ribofuranose (77). Asolution of 31 (12.5 g) in pyridine at 0° C. was treated withp-toluenesulphonyl chloride and the reaction was continued at roomtemperature for 14-16 h. Removal of pyridine, extraction with methylenechloride and saturated bicarbonate solution afforded 77, 14 g, (80%).Homogeneous by TLC.

1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-tosyl-D-ribofuranose (78).Hydrolysis of 77 (14 g) was done in 75% acetic acid at 65° C. for 18 h.The solvent was removed under pressure and the residue was treated withethanol (3×100), toluene (3×50) and anhydrous pyridine (2×50). (Thiscompound 78 crystallised from petroleum ether as fine white solid.) Theresidue was taken in dry pyridine and treated with acetic anhydride atroom temperature for 8 h. Extraction with ethyl acetate and saturatedbicarbonate followed by washing with brine afforded 78 as a mixture of aand 13 anomers, 12 g, (83%). A direct comparison with an authenticsample of 78 (TLC, HPLC, NMR) confirmed its identity and purity.

Example 105

1-(2-O-Acetyl-3,5-di-O-benzyl-4-C-(p-toulenesulfonyloxymethyl)-β-D-ribofuranosyl)thymine(79). To a stirred solution of the anomeric mixture 78 (12.8 g, 21.4mmol) and thymine (5.38 g, 42.7 mmol) in anhydrous acetonitrile (182cm³) was added N,O-bis(trimethylsilyl)acetamide (31.68 ml, 128.23 mmol).The reaction mixture was stirred for 1 h at room temperature, andstirring was continued at 60° C. for 1.5 h. After cooling to 0° C.,trimethylsilyl triflate (6.57 ml, 30.33 mmol) was added dropwise, andthe mixture was stirred at 60° C. for 10 h. The reaction mixture wasneutralised with an ice-cold saturated aqueous solution of sodiumhydrogen carbonate (90 mL). The reaction mixture was filtered, and thefiltrate was concentrated under reduced pressure to half volume.Extraction was performed using dichloromethane (4×200 cm³). The combinedorganic phase was washed with saturated aqueous solutions of sodiumhydrogen carbonate (3×150 cm³) and brine (3×150 ml), dried (Na₂SO₄),filtered and evaporated under reduced pressure. The residue was purifiedby silica gel column chromatography using dichloromethane:methanol (99:1to 98:2, v/v) as eluent to give nucleoside 79 as a white solid material(13.1 g, 92%). δ_(H) (CDCl₃) 9.04 (s, 1H, NH), 7.73-7.19 (15H, m, 6-H,aromatic), 5.94 (1H, d, J 5.5, 1′-H), 5.37 (1H, d, J 5.6, 2′-H),4.57-4.40 (5H, m, 3′-H, 5′-H_(a), 5′-H_(b, Bn),) 4.14 (2H, s, Bn), 3.75(1H, d, J 10.2, 1″-H_(a)), 3.57 (1H, d, J 10.2, 1″-H_(b)), 2.41 (3H, s,CH₃C₆H₅), 2.02 (3H, s, COCH₃), 1.54 (3H, s, CH₃). δ_(C) (CDCl₃) 169.8(C═O), 163.5 (C-4), 150.2 (C-2), 145.0, 136.8, 135.6, 132.1, 129.7,128.5, 128.0, 127.9, 127.8, 127.5 (aromatic), 113.5 (C-5), 86.8, 85.3,77.6, 74.6, 74.3, 73.6, 70.8, 68.8 (Bn, C-1′, C-3′, C-2′, C-4′), 21.3(CH₃), 20.5 (COCH₃), 11.8 (CH₃). FAB-MS m/z 665 [M+H]⁺ (Found C, 61.2;H, 5.3; N, 4.1; S, 4.7, C₃₄H₃₆O₁₀N₂S requires C, 61.4; H, 5.4; N, 4.2;S, 4.8).

Example 106

1-(3,5-Di-O-benzyl-4-C-(p-toulenesulfonyloxymethyl)-β-D-ribofuranosyl)-thymine(80). Nucleoside 79 (13.1 g, 19.7 mmol) was dissolved in a solution ofammonia in methanol (200 cm³, prepared by diluting saturated methanolicammonia with an equal volume of methanol) and stirred at roomtemperature for 4 h. The reaction mixture was subsequently evaporated,and the residue was dissolved in dichloromethane (400 cm³). The organicphase was washed with brine (3×150 cm³), dried (Na₂SO₄), filtered andevaporated under reduced pressure. The residue was purified by silicagel column chromatography using dichloromethane:methanol (99.5:0.5, v/v)as eluent to give nucleoside 80 as a white solid material (10.7 g, 87%).δ_(H) (CDCl₃) 9.66 (s, 1H, NH), 7.71-7.21 (15H, m, 6-H, aromatic), 5.72(1H, d, J 5.1, 1′-H), 4.75, 4.55 (2H, each d, J 11.5, Bn), 4.51 (2H, s,Bn), 4.37 (1H, t, J 5.4, 2*-H), 4.30-4.12 (3H, m, 3′-H, Bn), 3.76 (1H,d, J 10.2, 1″-H_(a)), 3.59 (1H, d, J 10.2, 1″-H_(b)), 2.39 (3H, s,CH₃C₆H₅), 1.48 (3H, s, CH₃). δ_(C) (CDCl₃) 163.8 (C-4), 150.9 (C-2),145.0, 137.0, 136.9, 135.9, 132.3, 129.8, 128.7, 128.6, 128.2, 128.1,128.0, 127.6 (aromatic), 111.0 (C-5), 89.6, 85.3, 78.4, 74.5, 73.8,71.1, 69.7, (Bn, C-1′, C-3′, C-2′, C-4′, C-1″), 21.6 (CH₃), 12.0 (CH₃).FAB-MS m/z 623 [M+H]⁺ (Found C, 61.5; H, 5.2; N, 4.4; S, 5.2,C₃₂H₃₄O₉N₂S requires C, 61.7; H, 5.4; N, 4.5; S, 5.1).

Example 107

(1S,3R,4R,7S)-7-Benzyloxy-1-benzoyloxymethyl-3-(thymin-1-yl)-2,5-dioxabi-cyclo[2.2.1]heptane(36). To a stirred solution of nucleoside 80 (10.65 g, 17.1 mmol) inanhydrous DMF (150 cm³) was added a 60% suspension of sodium hydride inmineral oil (0.9 g, 22.2 mmol) in small portions at 0° C. The mixturewas stirred at 0° C. for 15 h whereupon additional 60% sodium hydride(0.205 g, 5.12 mmol) was added, and the reaction mixture was stirred foradditional 22 h at 0° C. Methanol (20 cm³) was added and the reactionmixture was subsequently concentrated under reduced pressure to halfvolume. Ice-cold H₂O (300 cm³) was added and extraction was performedwith dichloromethane (5×150 cm³). The combined organic phase was washedwith saturated aqueous solutions of sodium hydrogen carbonate (3×40 cm³)and brine (3×40 cm³), dried (Na₂SO₄), filtered and evaporated underreduced pressure. The residue was purified by silica gel columnchromatography using dichloromethane:methanol (99.5:0.5, v/v) as eluentto give nucleoside 36 as a white solid material (7.1 g, 92%). Spectraldata were in accordance with data given earlier for 36 (Found C, 66.2;H, 5.8; N, 6.1; C₂₅H₂₆N₂O₆ requires C, 66.6; H, 5.8; N, 6.2).

Example 108

3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-methanesulfonyloxymethyl-α-D-ribofuranose(200). To a stirred solution of furanose 31 (2.16 g, 5.39 mmol) inanhydrous pyridine (3 mL) at 0° C. was added dropwise methanesulfonylchloride (0.61 mL, 16.0 mmol). The reaction mixture was stirred for 20min at room temperature, quenched with ice-cold water (300 mL) andextracted with dichloromethane (2×300 mL). The combined extracts werewashed with saturated aqueous sodium hydrogen carbonate (300 mL) andthen dried (MgSO₄). The solvent was removed by distillation underreduced pressure and the residue was purified by chromatography oversilica gel with dichloromethane as eluent to give the product 200 as aclear oil (2.55 g, 99%); ¹H NMR (CDCl₃): δ 7.37-7.24 (10H, m, Bn), 5.78(1H, d, J 3.8 Hz, H-1), 4.85 (1H, d, J 11.7 Hz, Bn), 4.73 (1H, d, J 11.9Hz, Bn), 4.64 (1H, dd, J 4.0, 5.3 Hz, H-2), 4.54 (1H, d, J 11.9 Hz,H-5′), 4.52 (1H, d, J 11.9 Hz, Bn), 4.46 (1H, d, J 11.9 Hz, H-5′), 4.41(1H, d, J 11.8 Hz, Bn), 3.60 (1H, d, J 10.4 Hz, H-5), 3.50 (1H, d, J10.5 Hz, H-5), 3.06 (3H, s, SO₂CH₃), 1.68 (3H, s, CH₃), 1.34 (3H, s,CH₃); ¹³C NMR (CDCl₃): δ 137.79, 137.31, 128.54, 128.48, 128.16, 128.01,127.87, 127.79 (Bn), 113.66 (C(CH₃)₂)_(,) 104.46 (C-1), 84.88 (C-4),78.48, 78.41 (C-2, C-3), 73.65, 72.63, 70.78, 70.16 (Bn, C-5, C-5′),37.84 (SO₂CH₃), 26.20 (CH₃), 25.69 (CH₃); MS FAB: 501 (M+Na, 100%).Found: C, 60.37; H, 6.29; S, 6.53; C₂₄H₃₀O₈S requires C, 60.24; H, 6.32;S, 6.70%.

Example 109

Methyl 3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-α-D-ribofuranoside(201). A solution of furanose 200 (1.133 g, 2.37 mmol) in methanolichydrochloric acid (20% w/w, 31.7 mL) and water (4.4 mL) was stirred atroom temperature for 2 h. After neutralisation with sodium hydrogencarbonate (s), the solution was extracted with dichloromethane (2×150mL). The combined extracts were washed with water (150 mL) and thendried (MgSO₄). The solvent was removed by distillation under reducedpressure and the residue purified by chromatography over silica gel withdichloromethane:methanol (99:1) as eluent to give the product 201(β:α˜2:1) as a clear oil (1.018 g, 95%); ¹H NMR (CDCl₃): δ 7.39-7.22 (m,Bn), 4.86 (br s, Bn), 4.69-3.99 (m, Bn, H-5′, H-1, H-2, H-3), 3.68 (d, J8.9 Hz, H-5 β), 3.51 (d, J 9.8 Hz, H-5 α), 3.46 (s, OCH₃ α), 3.34 (d, J9.1 Hz, H-5 β), 3.32 (d, J 9.7 Hz, H-5α), 3.28 (s, OCH₃ β), 2.97 (3 H,s, SO₂CH₃ β), 2.93 (3H, s, SO₂CH₃ α); ¹³C NMR (CDCl₃): δ 137.74, 136.98,128.70, 128.64, 128.58, 128.56, 128.37, 128.21, 128.15, 128.09, 127.98,127.86, 127.83 (Bn), 107.54 (C-1 β), 103.39 (C-1α), 84.65, 83.18, 81.90,78.87 (C-4, C-3), 75.04, 74.07, 73.73, 73.70, 73.38, 72.56, 72.11,70.85, 70.55, 70.20 (C-2, Bn, C-5, C-5′), 55.90 (OCH₃ α), 54.96 (OCH₃β), 37.18 (SO₂CH₃ β), 37.07 (SO₂CH₃ α); MS FAB: 475 (M+Na, 25%). Found:C, 58.40; H, 6.33; C₂₄H₃₀O₈S requires C, 58.39; H, 6.24%.

Example 110

(3R)- and (3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo[2.2.1]heptane(202 and 203). A solution of 201 (3.32 g, 7.34 mmol) in anhydrous DMF(25 mL) was stirred at 0° C. and a 60% oil dispersion of sodium hydride(700 mg, 16.9 mmol) was added. The mixture was stirred at roomtemperature for 90 min, quenched with water (300 mL) and extracted withdiethyl ether (2×300 mL). The combined extract was washed with water(200 mL) and dried (MgSO₄). The solvent was removed under reducedpressure and the residue was purified by chromatography over silica gelwith dichloromethane as eluent to give the two products 202 and 203 asclear oils (1.571 g, 60% and 0.777 g, 30% respectively).(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo[2.2.1]heptane(202). ¹H NMR (CDCl₃): δ 7.36-7.26 (10H, m, Bn), 4.81 (1H, s, H-1), 4.65(1H, d, J 11.9 Hz, Bn), 4.61 (2H, s, Bn), 4.56 (1H, d, J 11.9 Hz, Bn),4.11 (1H, s, H-2), 4.09 (1H, s, H-3), 4.01 (1H, d, J 7.5 Hz, H-5′),3.80-3.77 (3H, m, H-5′, H-5), 3.39 (3H, s, OCH₃); ¹³C NMR (CDCl₃): δ138.05, 137.36, 128.47, 128.44, 127.88, 127.73, 127.63 (Bn), 104.97(C-1), 85.13 (C-4), 79.16 (C-3), 77.18 (C-2), 73.64 (Bn), 72.26, 72.10(Bn, C-5′), 66.50 (C-5), 55.34 (OCH₃); MS FAB: 379 (M+Na, 28%). Found:C, 70.55; H, 6.97; C₂₁H₂₄O₅ requires C, 70.77; H, 6.79%.(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo-[2.2.1]heptane(203). ¹1-1 NMR (CDCl₃): δ 7.36-7.26 (10H, m, Bn), 5.00 (1H, s, H-1),4.67-4.54 (4H, m, Bn), 4.18 (1H, s, H-2), 3.99 (1H, s, H-3), 3.99-3.90(2H, m, H-5′), 3.75-3.68 (2H, m, H-5), 3.49 (3H, s, OCH₃); ¹³C NMR(CDCl₃): δ 137.83, 137.53, 128.51, 128.48, 127.96, 127.82, 127.71,127.62 (Bn), 104.05 (C-1), 88.44 (C-4), 79.54 (C-3), 77.16 (C-2), 73.68(Bn), 72.61 (C-5′), 72.24 (Bn), 65.73 (C-5), 56.20 (OCH₃); MS FAB: 379(M+Na, 100%).

Example 111

(1R,2S,3S)-2-Benzyloxy-3-benzyloxymethyl-1-(methoxy(thymin-1-yOmethyl)-3-trimethylsilyloxytetrahydrofuran(204). A solution of 202 (216 mg, 0.606 mmol) and thymine (153 mg, 1.22mmol) in anhydrous acetonitrile (9.3 mL) was added BSA(N,O-bis(trimethylsilyl)acetamide, 0.90 mL, 3.6 mmol) and stirred underreflux for 15 min. The solution was cooled to 0° C. and trimethylsilyltriflate (0.153 mL, 0.777 mmol) was added dropwise. After stirring atroom temperature for 18 h and at 60° C. for 24 h, the reaction wasquenched with a saturated aqueous solution of sodium hydrogen carbonate(20 mL), and extraction was performed using dichloromethane (2×50 mL).The combined extract was washed with a saturated aqueous solution ofsodium hydrogen carbonate (50 mL) and dried (MgSO₄). The solvent wasremoved under reduced pressure and the residue was purified bychromatography over silica gel with dichloromethane:methanol (98:2) aseluent to give the product 204 (mixture of diastereomers ˜1.7:1) as asolid (196 mg, 67%). ¹H NMR (CDCl₃): δ 7.36-7.14 (m, Bn, H-6), 5.77 (1H,d, J 7.9 Hz, H-1′), 5.57 (1 H, d, J 5.8 Hz, H-1′), 4.68-4.43 (m, Bn,H-2′), 4.12-3.68 (m, H-5′, H-5′, H-3′), 3.32 (s, OCH₃), 3.24 (s, OCH₃),1.93 (d, J 0.9 Hz, CH₃), 1.86 (d, J 1.1 Hz, CH₃), 0.14 (s, Si(CH₃)₃),0.12 (s, Si(CH₃)₃); ¹³C NMR (CDCl₃): δ 163.68, 163.55 (C-4), 151.58,151.07 (C-2), 137.84, 137.74, 137.32 (Bn), 135.93, 135.10 (C-6), 128.57,128.42, 128.41, 128.10, 127.95, 127.85, 127.77, 127.74 (Bn), 111.38,111.01 (C-5), 86.89, 85.61, 85.40, 84.72, 83.40, 83.31, 82.10 (C-1′,C-2′, C-3′, C-4′), 75.20, 73.98, 73.62, 73.59, 72.55, 72.13, 71.04,70.74 (Bn, C-5′, C-5″), 56.82, 56.54 (OCH₃), 12.47, 12.38 (CH₃), 1.72,1.69 (Si(CH₃)₃); MS FAB: 555 (M+H, 65%), 577 (M+Na, 70%). Found: C,62.76; H, 6.88; N, 4.94; C₂₉H₃₈N₂O₇Si requires C, 62.79; H, 6.90; N,5.05%.

Example 112

(1R,2S,3S)-2-Benzyloxy-3-benzyloxymethyl-1-(methoxy(6-N-benzoyladenin-9-yl)methyl)-3-trimethylsilyloxytetrahydrofuran(205). A solution of 202 (240 mg, 0.673 mmol) and 6-N-benzoyladenine(301 mg, 1.26 mmol) in anhydrous acetonitrile (8.2 mL) was added BSA(0.67 mL, 2.7 mmol) and stirred at room temperature for 1 h. Thesolution was cooled to 0° C. and trimethylsilyl triflate (0.25 mL, 1.33mmol) was added dropwise. After stiffing at 65° C. for 18 h, thereaction was quenched with a saturated aqueous solution of sodiumhydrogen carbonate (50 mL), extracted with dichloromethane (2×50 mL).The combined extract was dried (MgSO₄). The solvent was removed underreduced pressure and the residue was purified by chromatography oversilica gel with dichloromethane:methanol (98:2) as eluent to give theproduct 205 (mixture of diastereomers ˜1.8:1) as a solid (185 mg, 41%).¹H NMR (CDCl₃): δ 8.78 (s, H-8), 8.21 (s, H-2), 8.17 (s, H-2), 8.03-8.00(m, Bz), 7.61-7.49 (m, Bz), 7.36-7.23 (m, Bn), 7.07-7.04 (m, Bz), 5.85(1H, d, J 7.9 Hz, H-1′), 5.76 (1H, d, J 6.0 Hz, H-1′), 4.74-4.40 (m, Bn,H-2′), 4.22-3.62 (m, H-5′, H-5′, H-3′), 3.33 (s, OCH₃), 3.24 (s, OCH₃),0.15 (s, Si(CH₃)₃), 0.14 (s, Si(CH₃)₃); ¹³C NMR (CDCl₃): δ 164.68(HNC═O), 153.17, 152.99 (C-6), 149.47 (C-2), 141.82, 141.66 (C-8),137.74, 137.71, 137.65 (Bn), 133.87, 132.87, 132.78 (Bz), 128.97,128.93, 128.45, 128.42, 128.38, 128.14, 127.97, 127.88, 127.82, 127.78(Bn, Bz), 123.66, 122.85 (C-5), 86.41, 86.23, 85.70, 85.24, 84.78,83.73, 83.58, 82.79 (C-1′, C-2′, C-3′, C-4′), 75.32, 74.55, 73.61,72.18, 71.98, 70.85, 70.59 (Bn, C-5′, C-5″), 57.23, 57.04 (OCH₃), 1.78(Si(CH₃)₃); MS FAB: 668 (M+H, 50%), 690 (M+Na, 100%). Found: C, 64.07;H, 6.01; N, 9.94; C₂₉H₃₈N₂O₇Si,0.5H₂O requires C, 63.88; H, 6.25; N,10.34%.

Example 113

(1R,2R,3R)-2-Benzyloxy-3-benzyloxymethyl-3-hydroxytetrahydro-furfural(206). A solution of 202/203 (252 mg, 0.707 mmol) in 80% acetic acid(3.8 mL) was stirred at 90° C. for 2 h whereupon the solvent was removedby distillation under reduced pressure. The residue was coevaporated intoluene (3×10 mL) to give the product 206 as an oil (242 mg, 100%). ¹HNMR (CDCl₃): δ 9.66 (1H, d, J 0.8 Hz, H-1), 7.36-7.25 (10 H, m, Bn),4.68 (1H, d, J 11.9 Hz, Bn), 4.60-4.39 (5H, m, Bn, H-2, H-3), 3.98-3.92(2H, m, H-5), 3.85 (1H, d, J 9.3 Hz, H-5′), 3.52 (1H, d, J 9.2 Hz,H-5′); ¹³C NMR (CDCl₃): δ 203.64 (C-1), 137.39, 137.19, 128.61, 128.54,128.29, 128.12, 127.87, 127.83 (Bn), 87.17, 87.05 (C-4, C-2), 80.98(C-3), 75.00, 73.70, 71.86 (Bn, C-5′), 67.84 (C-5); MS FAB: 707 (2×M+Na,100%).

Example 114

(1S,3S,4R,7S)-3-Acetoxy-7-benzyloxy-1-benzyloxymethyl-2,5-dioxabicyclo-[2.2.1]heptane(207). To a stirred solution of 206 (230 mg, 0.672 mmol) in anhydrouspyridine (2.0 mL) was added acetic anhydride (0.18 mL, 1.91 mmol). Thereaction mixture was stirred for 23 h at room temperature, water (0.13mL) was added, and the solvent was removed by distillation under reducedpressure. The residue was coevaporated in toluene (3×10 mL) and purifiedby chromatography over silica gel with dichloromethane:methanol (99:1)as eluent to give the product 207 as an clear oil (56.7 mg, 23%); ¹H NMR(CDCl₃): δ 7.38-7.26 (10H, m, Bn), 6.00 (1H, s, H-1), 4.68 (1H, d, J12.0 Hz, Bn), 4.62 (1H, d, J 12.2 Hz, Bn), 4.60 (1H, d, J 12.4 Hz, Bn),4.56 (1H, d, J 12.2 Hz, Bn), 4.17 (1H, s, H-2), 4.14 (1H, s, H-3), 4.01(1H, d, J 7.7 Hz, H-5′), 3.81-3.78 (3H, m, H-5′, H-5), 20.06 (3H, s,COCH₃); ¹³C NMR (CDCl₃): δ 169.18 (C═O), 137.92, 137.48, 128.52, 128.45,128.03, 127.77, 127.73, 127.68 (Bn), 95.95 (C-1), 86.49 (C-4), 78.27,76.58 (C-3, C-2), 73.65 (Bn), 72.26, 71.96 (Bn, C-5′), 65.49 (C-5),20.98 (COCH₃); MS FAB: 407 (M+Na, 55%). Found: C, 68.80; H, 6.11;C₂₂H₂₄O₆ requires C, 68.74; H, 6.29%.

Example 115

(1S,3S,4R,7S)-3-(6-N-Benzoyladenin-9-yl)-7-benzyloxy-1-benzyloxymethyl-2,5-dioxabicyclo[2.2.1]heptane(208). A solution of furanose 207 (167 mg, 0.434 mmol) and6-N-benzoyladenine (194 mg, 0.813 mmol) in anhydrous acetonitrile (5.3mL) was added BSA (0.43 mL, 1.76 mmol) and stirred at room temperaturefor 1 h. The solution was cooled to 0° C. and trimethylsilyl triflate(0.16 mL, 0.86 mmol) was added dropwise. After stiffing at 65° C. for 2h, the reaction was quenched with a saturated aqueous solution of sodiumhydrogen carbonate (40 mL) and the mixture was extracted withdichloromethane (2×50 mL). The combined extract was dried (MgSO₄). Thesolvent was removed under reduced pressure and the residue was purifiedby chromatography over silica gel with dichloromethane:methanol (98:2)as eluent to give the product 208 as a solid (111 mg, 45%); ¹H NMR(CDCl₃): δ 8.82 (1H, s, H-8), 8.14 (1H, s, H-2), 7.59-7.26 (15H, m, Bz,Bn), 6.74 (1H, s, H-1′), 4.92 (1H, s, H-2′), 4.74-4.39 (4H, m, Bn), 4.42(1H, s, H-3′), 4.19-4.10 (2H, m, H-5″), 3.92 (1H, d, J 11.8 Hz, H-5′),3.88 (1H, d, J 11.5 Hz, H-5′); MS FAB: 564 (M+H, 100%).

Example 116

Methyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-D-ribofuranoside(209). To a stirred solution of 201 (687 mg, 1.52 mmol) in anhydrouspyridine (4 mL) at 0° C. was added dropwise acetic anhydride (0.43 mL,4.56 mmol). The reaction mixture was stirred for 2 days at roomtemperature, quenched with saturated aqueous sodium hydrogen carbonate(75 mL) and extracted with dichloromethane (150+75 mL). The combinedextract was dried (MgSO₄), the solvent was removed by distillation underreduced pressure and the residue was purified by chromatography oversilica gel with dichloromethane as eluent to give the product 209 as aclear oil (β:α˜3:1, 750 mg, 100%); MS FAB: 463 (M-OCH₃, 100%), 517(M+Na, 28%); Found: C, 58.53; H, 6.16; C₂₄H₃₀O₉S requires C, 58.29; H,6.11%. Methyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-β-D-ribofuranoside(209β). ¹H NMR (CDCl₃): δ 7.36-7.18 (10H, m, Bn), 5.27 (1H, d, J 4.9 Hz,H-2), 4.88 (1H, s, H-1), 4.55-4.44 (6H, m, H-5′, Bn), 4.35 (1H, d, J 5.0Hz, H-3), 3.73 (1H, d, J 9.2 Hz, H-5), 3.38 (1H, d, J 9.3 Hz, H-5), 3.30(3H, s, OCH₃), 2.95 (3H, s, SO₂CH₃), 2.11 (3H, s, OCCH₃); ¹³C NMR(CDCl₃): δ 169.91 (C═O), 137.83, 137.28, 128.49, 128.44, 127.99, 127.87,127.77 (Bn), 105.40 (C-1), 82.65, 81.05, 74.55, 73.62, 73.56, 71.86,70.22 (C-2, C-3, C-4, C-5, C-5′, Bn), 55.03 (OCH₃), 37.14 (SO₂CH₃),20.73 (OCCH₃). Methyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-α-D-ribofuranoside(209α). ¹H NMR (CDCl₃): δ 7.36-7.18 (10H, m, Bn), 5.09 (1H, d, J 4.5 Hz,H-1), 4.95 (1H, dd, J 4.5, 6.8 Hz, H-2), 4.65-4.44 (6H, m, H-5′, Bn),4.27 (1H, d, J 6.6 Hz, H-3), 3.49 (1H, d, J 9.9 Hz, H-5), 3.46 (3H, s,OCH₃), 3.36 (1H, d, J 9.9 Hz, H-5), 2.92 (3H, s, SO₂CH₃), 2.14 (3H, s,OCCH₃); ¹³C NMR (CDCl₃): δ 170.41 (C═O), 137.59, 137.28, 128.56, 128.51,128.49, 128.44, 127.98, 127.88 (Bn), 102.35 (C-1), 84.25, 77.53, 74.66,73.67, 72.12, 70.39, 70.28 (C-2, C-3, C-4, C-5, C-5′, Bn), 56.07 (OCH₃),36.94 (SO₂CH₃), 20.63 (OCCH₃).

Example 117

Phenyl2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-1-thio-β-D-ribofuranoside(210). Method a. A stirred solution of 209 (738 mg, 1.49 mmol) inanhydrous dichloromethane (6.4 mL) was added phenylthiotrimethylsilane(2.42 mL, 12.8 mmol) and cooled to 0° C. Trimethylsilyl triflate (0.67mL, 3.67 mmol) was added dropwise and the solution was stirred at roomtemperature for 4 h. The reaction was quenched with a saturated aqueoussolution of sodium hydrogen carbonate (100 mL) and extracted withdichloromethane (2×200 mL). The combined extract was dried (MgSO₄) andthe solvent removed by distillation under reduced pressure. The residuewas purified by chromatography over silica gel with dichloromethane aseluent to give the product 210 as a clear oil (564 mg, 66%) andunreacted starting material (191 mg, 26%); Method b. A stirred solutionof 211 (86 mg, 0.165 mmol) in anhydrous dichloromethane (0.49 mL) wasadded phenylthiotrimethylsilane (0.16 mL, 0.825 mmol) and cooled to 0°C. Trimethylsilyl triflate (0.037 mL, 0.206 mmol) was added and thesolution was stirred at room temperature for 2 h. The reaction wasquenched with a saturated aqueous solution of sodium hydrogen carbonate(15 mL) and the resulting mixture was extracted with dichloromethane(2×25 mL). The combined extract was dried (MgSO₄) and the solventremoved by distillation under reduced pressure. The residue was purifiedby chromatography over silica gel with dichloromethane as eluent to givethe product 210 as a clear oil (75 mg, 79%); ¹H NMR (CDCl₃): δ 7.47-7.19(15H, m, Bn, SPh), 5.48 (1H, d, J 3.6 Hz, H-2), 5.34 (1H, dd, J 3.7, 5.2Hz, H-1), 4.54-4.36 (7H, m, H-3, H-5′, Bn), 3.66 (1H, d, J 9.7 Hz, H-5),3.48 (1H, d, J 9.5 Hz, H-5), 2.89 (3H, s, SO₂CH₃), 2.09 (3H, s, OCCH₃);¹³C NMR (CDCl₃): δ 169.93 (C═O), 137.69, 137.08, 132.65, 132.45, 129.15,128.53, 128.52, 128.18, 128.14, 128.08, 127.91, 127.85 (Bn, SPh), 87.99,84.35, 80.34, 75.33, 74.20, 73.67, 70.83, 69.34 (C-1, C-2, C-3, C-4,C-5, C-5′, Bn), 37.27 (SO₂CH₃), 20.68 (OCCH₃); MS FAB: 463 (M-SPh,100%), 595 (M+Na, 24%); Found: C, 61.17; H, 5.55; C₂₉H₃₂O₈S₂ requires C,60.82; H, 5.63%.

Example 118

1,2-Di-O-acetyl-3,5-di-O-benzyl-4-C-methanesulphonyloxymethyl-D-ribofuranose(211). A solution of 201 (150 mg; 0.313 mmol) in 80% aqueous acetic acid(1.5 mL) was stirred at 90° C. for 3 h. The solvent was removed bydistillation under reduced pressure and the residue was coevaporated inethanol (3×5 mL), toluene (3×5 mL) and pyridine (2×5 mL). The residuewas redissolved in anhydrous pyridine (0.62 mL) and added aceticanhydride (0.47 mL) and the solution was stirred at room temperature for16 h. The reaction was quenched with water (50 mL) and the resultingmixture extracted with dichloromethane (2×50 mL). The combined extractwas washed with an aqueous saturated solution of sodium hydrogencarbonate (50 mL) and dried (MgSO₄). The solvent was evaporated and theresidue purified on column chromatography over silica gel withdichloromethane as eluent to give the product 211 as an oil (99 mg,60%); ¹H NMR (CDCl₃): δ 7.39-7.21 (m, Bn), 6.38 (d, J 4.6 Hz, H-1β),6.15 (s, H-1α), 5.35 (d, J 4.9 Hz, H-2α), 5.17 (dd, J 6.3, 4.9 Hz, H-2β), 4.69-4.23 (m, H-3, Bn), 3.64 (d, J 9.7 Hz, H-5 α), 3.52 (d, J 10.1Hz, H-2 β), 3.45 (d, J 9.7 Hz, H-5 α), 3.39 (d, J 9.9 Hz, H-2 β), 2.99(s, SO₂CH₃ α), 2.96 (s, SO₂CH₃ β), 2.14, 2.13, 2.06, 1.90 (4×s, COCH₃);¹³C NMR (CDCl₃): δ 169.68, 169.00 (C═O), 137.68, 137.05, 128.60, 128.55,128.50, 128.21, 128.12, 128.04, 127.94, 127.82, 127.79 (Bn), 99.35 (C-1α), 94.24 (C-1 β), 86.36 (C-4 β), 84.28 (C-4α), 79.15, 77.47, 74.58,74.06, 73.73, 73.56, 71.67, 70.57, 70.19, 69.84 (Bn, C-2, C-3, C-5,C-5′), 37.61 (SO₂CH₃ β), 37.48 (SO₂CH₃ α), 21.07, 20.74, 20.63, 20.39(COCH₃); MS FAB: 545 (M+Na, 13%). Found: C, 57.70; H, 5.56; C₂₅H₃₀O₁₀Srequires C, 57.46; H, 5.79%.

Example 119

(3R)- and(3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo[2.2.1]heptane(212). A solution of 210 (553 mg, 0.966 mmol) in methanol saturated withammonia (35 mL) was stirred at room temperature for 2 h whereupon thesolvent removed by distillation under reduced pressure. The residue wasredissolved in anhydrous DMF (3.5 mL) and the solution stirred at 0° C.A 60% suspension of sodium hydride (118 mg, 2.88 mmol) was added and themixture stirred at room temperature for 12 h. The reaction was quenchedwith a saturated aqueous solution of sodium hydrogen carbonate (100 mL)and the resulting mixture was extracted with dichloromethane (2×100 mL).The combined extract was dried (MgSO₄) and the solvent was removed bydistillation under reduced pressure. The residue was purified bychromatography over silica gel with dichloromethane as eluent to givethe product 212 as a clear oil (404 mg, 96%). MS FAB: 435 (M+H, 35%),457 (M+Na, 16%); Found: C, 71.76; H, 6.18; C₂₆H₂₆O₄S requires C, 71.86;H, 6.03%.(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo[2.2.1]heptane(212β). ¹H NMR (CDCl₃): δ 7.46-7.26 (15H, m, Bn, SPh), 5.35 (1H, s,H-1), 4.68-4.56 (4H, m, Bn), 4.31 (1H, s, H-2), 4.10 (1H, s, H-3), 4.09(1H, d, J 7.3 Hz, H-5′), 3.93 (1H, d, J 7.8 Hz, H-5′), 3.79 (2H, m,H-5); ¹³C NMR (CDCl₃): δ 138.03, 137.45, 133.42, 132.36, 129.19, 128.55,128.46, 128.05, 127.84, 127.83, 127.76 (Bn, SPh), 89.96 (C-1), 87.18(C-4), 79.71 (C-2), 79.40 (C-3), 73.64 (Bn), 73.23 (C-5′), 72.30 (Bn),66.31 (C-5).(1S,3S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo[2.2.1]-heptane(212a). ¹H NMR (CDCl₃): δ 7.52-7.19 (15H, m, Bn, SPh), 5.52 (1H, s,H-1), 4.70-4.50 (4H, m, Bn), 4.41 (1H, s, H-2), 4.18 (1H, d, J 7.8 Hz,H-5′), 4.08 (1H, d, J 8.4 Hz, H-5′), 4.07 (1H, s, H-3), 3.78 (1H, d, J11.3 Hz, H-5), 3.72 (1H, d, J 11.5 Hz, H-5); ¹³C NMR (CDCl₃): δ 137.89,137.46, 135.29, 130.93, 129.13, 128.99, 128.57, 128.48, 127.81, 127.76,127.58, 126.95 (Bn, SPh), 91.87 (C-1), 88.59 (C-4), 80.07, 79.14 (C-2,C-3), 73.65, 73.40, 72.04 (Bn, C-5′), 65.62 (C-5).

Example 120

(3R)- and(3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(36+213). Thymine (175 mg, 1.38 mmol) was stirred inhexamethyldisilazane (6.8 mL) at reflux and ammonium sulphate (5 mg) wasadded. After stirring for 16 h, the clear solution was cooled to 40° C.and the solvent was removed by distillation under reduced pressure. Tothe residue was added a solution of 212 (201 mg, 0.463 mmol) inanhydrous dichloromethane (4.6 mL) and 4 Å molecular sieves (180 mg).After stirring at room temperature for 10 min, NBS (107 mg, 0.602 mmol)was added and the mixture stirred for another 30 min. The reaction wasquenched with a saturated aqueous solution of sodium thiosulphate (25mL) and the resulting mixture was extracted with dichloromethane (2×50mL). The combined extract was dried (MgSO₄) and evaporated, and theresidue was purified on column chromatography over silica gel withdichloromethane:methanol (97:3) as eluent to give the product 36+213 andas an anomeric mixture (β:α˜1:2) (127 mg, 61%); ¹H NMR (CDCl₃): δ 7.49(d, J 0.9 Hz, H-6 β), 7.46 (d, J 1.0 Hz, H-6 cc), 7.39-7.25 (m, Bn),5.94 (s, H-1′ cc), 5.64 (s, H-1′ β), 4.71-4.50 (m, Bn, H-2′), 4.23 (s,H-3′ α), 4.16 (d, J 8.6 Hz, H-5″α), 4.09-3.78 (m, H-5′, H-5″, H-3′β),1.94 (d, J 0.9 Hz, CH₃ α), 1.62 (d, J 1.2 Hz, CH₃ β); MS FAB: 551 (M+H,96%).

Example 121

(3R)- and(3S)-(1S,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane(37+214). A solution of 36+213 (175 mg, 0.39 mmol) in ethanol (2.7 mL)was stirred at room temperature and 20% palladium hydroxide over carbon(50 mg) was added. The mixture was degassed several times with argon andplaced under a hydrogen atmosphere. After stirring for 18 h, the mixturewas purified on column chromatography over silica gel withdichloromethane:methanol (95:5) as eluent to give a mixture of 37 and214 (1:1.2) (26 mg, 25%); ¹H NMR (CD₃OD): δ 7.78 (d, J 1.3 Hz, H-6 α),7.73 (d, J 1.2 Hz, H-6 β), 5.88 (s, H-1′ α), 5.53 (s, H-1′ β), 4.38 (s,H-2′ α), 4.34 (s, H-3′ α), 4.26 (s, H-2′ β), 4.08-3.69 (m, H-5′, H-5″,H-3′β), 1.92 (d, J 1.2 Hz, CH₃ cc), 1.88 (d, J 1.1 Hz, CH₃ β); ¹³C NMR(CD₃OD): δ 138.00 (C-6 α), 136.96 (C-6 β), 110.80 (C-5 β), 110.08 (C-5α), 92.49, 89.01 (C-4′, C-1′ α), 90.46, 88.37 (C-4′, C-1′ β), 80.89,74.27, 73.34 (C-2′, C-3′, C-5′ α), 80.59, 72.47, 70.39 (C-2′, C-3′, C-5′β), 59.29 (C-5″ α), 57.61 (C-5″ β), 12.52 (CH₃ α), 12.39 (CH₃ β); MS EI:270 (M⁺, 100%).

Preparation of LNA Phosphoramidites Example 122

4-N-Benzoyl-LNA-C[(1R,3R,4R,7S)-3-(4-N-benzoylcytosine-1-yl)-1-(hydroxymethyl)-7-hydroxy-2,5-dioxabicyclo{2.2.1}heptane]. LNA-C (formula Z) was taken in absolute ethanol andheated at reflux. To the refluxing solution, benzoic anhydride (2equivalents) was added and the reaction was followed by HPLC (Eluant:20% acetonitrile in 0.1M TEAA, pH 7.0, flow rate: 1 ml/min., NovapakC-18 analytical column). Additional anhydride was added at 0.5-2 hintervals till no more increase in product was observed by HPLC.Reaction mixture was concentrated on rotavap. Residue was repeatedlywashed with ether, filtered and dried to give an off white solid. Yield:45%.

General method for dimethoxytritylation of base protected LNAnucleosides (LNA-C^(Bz), LNA-T, LNA-G^(iBu), LNA-A^(Bz)). Base protectedLNA-nucleoside was coevaporated with pyridine (2×) and was stirred withdimethoxytrityl chloride (1.5 equivalents) in pyridine (˜10 ml/g ofnucleoside). The reaction was followed by HPLC (50% acetonitrile in 0.1MTEAA, pH 7.0, for 5 min., 50-100% acetonitrile in 10 min. and 100%acetonitrile for 5 min., flow rate: 1 ml/min., Novapak C-18 column).When >95% of the starting material had reacted, reaction mixture wascooled in ice. Reaction was quenched by addition of cold saturatedNaHCO₃ (˜15 ml×vol. of pyridine). The mixture was extracted withdichloromethane (3×half the vol. of sodium bicarbonate). Organicextractions were combined, dried over anhydrous sodium sulfate, filteredand concentrated on rotavap. Residue was dried in vacuo and purified bysilica gel chromatography using 0.5% pyridine and 0-2% methanol indichloromethane as eluant. Fractions containing pure products werecombined and concentrated on rotavap. Residue was coevaporated withanhydrous acetonitrile (3×) and dried in vacuo.

General method for phosphitylation of protected LNA nucleosides. Baseprotected dimethoxytrityl-LNA nucleoside was coevaporated with anhydrousdichloromethane (2×) and was taken in anhydrous dichloromethane (10 ml/gof nucleoside for A, G & T and ˜30 ml/g for C). To thisbis(diisopropylamino)(2-cyanoethyl)phosphite (1.05-1.10 equivalent),followed by tetrazole (0.95 equivalent) were added. Mixture was stirredat room temperature and reaction was followed by HPLC (70% acetonitrilein 0.1M TEAA, pH 7, 2 min., 70-100% acetonitrile in 8 min., and 100%acetonitrile in 5 min., flow rate: 1 ml/min., Novapak C-18 column). Oncethe reaction had proceeded to >90% and no more increase in amiditeformation was observed upon further stirring, the mixture was cooled inice. It was diluted with dichloromethane (˜15-20 times the originalvolume) and washed with cold saturated sodium bicarbonate (2×) followedby cold brine (1×). Organic layer was dried over anhydrous sodiumsulfate, filtered and concentrated on rotavap. Residue was coevaporatedwith anhydrous acetonitrile (3×) and dried in vacuo overnight. HPLCpurity ranged from 93-98%.

Preparation of LNA nucleoside 5′-triphosphates Example 123

Synthesis of LNA nucleoside 5′-triphosphates. (Tetrahedron Letters 1988,29 4525). In a 13×100 mm polypropylene tube, nucleosides 37, 44, 51,4-N-benzoylated 57A or 61B (93.8 μmol) was suspended in 1 mL pyridine(dried by CaH₂). The solution was evaporated in a speedvac, under highvacuum, to dryness. The residue was twice resuspended in acetonitrile(dried by CaH₂) and evaporated to dryness. The nucleoside was suspendedin 313 μL trimethyl phosphate (dried by 4 Å molecular sieves), to which30.1 mg Proton Sponge™ (1.5 equivalents) were added. The mixture wassealed, vortexed, and cooled to 0° C. POCl₃ (9.8 μL, 1.1 equivalent) wasadded with vortexing. The reaction was allowed to proceed at 0° C. for2.5 hours. During this interval, 469 μmols sodium pyrophosphate (5equivalents) were dissolved in 5 mL water and passed through 5 mL Dow50H⁺ ion exchange resin. When the effluent turned acidic, it wascollected in 220 μL tributylamine and evaporated to a syrup. The TBApyrophosphate was coevaporated three times with dry acetonitrile.Finally, the dried pyrophosphate was dissolved in 1.3 mL DMF (4Asieves). After 2.5 hours reaction time, the TBA pyrophosphate and 130 μLtributylamine were added to the nucleoside solution with vigorousvortexing. After 1 minute, the reaction was quenched by adding 3 mL 0.1M triethylammonium acetate, pH 7.5. Assay by Mono Q chromatographyshowed 49% nucleoside 5′-triphosphate. The reaction mixture was dilutedto 100 mL with water and adsorbed onto a Q Sepharose ion exchangecolumn, washed with water, and eluted with a linear gradient of 0 to 700mM NaCl in 5 mM sodium phosphate, pH 7.5. Fractions containingtriphosphate were assayed by Mono Q ion exchange chromatography.Fractions containing triphosphate were pooled and concentrated to thepoint of NaCl saturation. The product was desalted on a C₁₈ cartridge.The triphospate was quantitated by UV spectroscopy and adjusted to 10 mMsolution. Yields were 17-44%. LNA nucleosides prepared by this methodwere, U, T, A, G, and C.

Preparation of LNA Modified Oligonucleotides Example 124

Synthesis of oligonucleotides containing LNAs of formula V, X, Y andZ^(T), Z^(U), Z^(G), Z^(C), Z^(A), Z^(MeC). The bicyclic nucleoside3′-O-phosphoramidite analogues 8, 19, 30, 39, 46, 53, 57D, 61D, and 66as well as commercial 3′-O-phosphoramidites were used to synthesiseexample LNA oligonucleotides of the invention (0.2 to 5 μmol scale)containing one or more of the LNAs of types V, X, Y and Z^(T), Z^(U),Z^(G), Z^(C), Z^(A) and Z^(MeC). The purity and composition of thesynthesised LNA oligonucleotides was verified by capillary gelelectrophoresis, and/or HPLC and/or MALDI-MS. In general, satisfactorycoupling efficiencies were obtained for all the monomers. The bestcoupling efficiencies (˜95-100%) were obtained for LNAs 39, 46, 53, 57D,61D, and 66 (leading to LNA monomers of formula Z) giving verysatisfactory results when synthesising fully modified LNAoligonucleotides or when incorporating LNAs in otherwise unmodified DNAor RNA stands or LNAs into an all-phosphorothioate oligonucleotide. LNAoligonucleotides were dissolved in pure water and the concentrationdetermined as OD₂₆₀. Solubilities in all cases were excellent. For plainDNA/RNA synthesis and partially modified LNA oligomers, a standard CPGsupport or a polystyrene support, was used. For the synthesis of fullymodified LNA oligomers (e.g. 5′-d(GTGATATGC)-3′), a BioGenex UniversialCPG Support (BioGenex, U.S.A.) was used, or LNA derivatised supportswere used.)

Example 125

Synthesis of phosphorothioate LNA oligonucleotides. Theall-phosphorothioate LNA (Table 7) was synthesised on an automated DNAsynthesiser using similar conditions as those described earlier (Example124). Beaucages' reagent was used as sulphurising agent. The stepwisecoupling yields were >98%. After completion of the syntheses,deprotection and cleavage from the solid support was effected usingconcentrated ammonia (55° C., 14 h).

Example 126

Synthesis of 2′-Thio-LNA oligonucleotides. The 2′-thio-LNAoligonucleotides (containing monomer U^(S) (formula Z (thio-variant) ofFIG. 2), FIG. 37, Table 8) were synthesised on an automated DNAsynthesiser using standard conditions (Example 124). The step-wisecoupling yield for amidite 76F was approximately 85% (12 min couplings;improved purity of amidite 76F is expected to result in increasedcoupling yield). After completion of the syntheses, deprotection andcleavage from the solid support was effected using concentrated ammonia(55° C., 8 h).

Example 127

Synthesis of 2′-Amino-LNA oligonucleotides. By procedures similar tothose described in Example 126, 2′-Amino-LNA oligonucleotides(containing monomer T^(NH) and monomer T^(NMe) (formula Z (aminovariants) of FIG. 2), FIGS. 35 and 36) was efficiently obtained on anautomated DNA synthesiser using amidites 74A and 74F (98% stepwisecoupling yields).

Example 128

Fluorescein-labeling of LNA oligomers. LNA oligomers (formula Z of FIG.2) AL16 (5′-d(TGTGTGAAATTGTTAT)-3′; LNA nucleotides in bold) and AL17(5′-d(ATAAAGTGTAAAG)-3′; LNA nucleotides in bold) were succesfullylabeled with fluorescein using the FluoroAmp T4 Kinase GreenOligonucleotide Labeling System as described by the manufacturer(Promega). Briefly, 16 nmol of either LNA-oligomer AL16 or AL17 was5′-thiophosphate labelled in a 50 μl reaction buffer containing T4kinase and γ-S-ATP. The reactions were incubated for 2 h at 37° C. Thethiophosphorylated LNA oligos were precipitated by the addition of 50 ofoligonucleotide precipitant (Promega) and 165 μl of ice cold (20° C.)95% ethanol. After centrifugation the pellets were washed once with 500μl of ice cold (−20° C.) 70% ethanol and redissolved in 25 μl of PBSEbuffer. Freshly prepared 5-maleimide-fluorescein solution (50 μg in 5 μlDMSO) were added to the thiophosphorylated LNA oligos and the reactionmixtures incubated at 68° C. for 30 min. Additional5-maleimide-fluorescein (50 μg in 5 μl DMSO) were added to each LNAoligo and the reaction mixtures incubated for an additional 60 min.After incubation 10 μl of oligonucleotide precipitant was added to eachreaction mixture followed by 180 μl ice-cold (20° C.) and 1000N,N-dimethylformamide. The fluorescein labeled LNA oligos were isolatedby centrifugation followed by aspiration of the supernatant. Thefluorescein labelled LNA-oligomers were purified by reversed-phase HPLCas follows: column Delta-Pack C-18, 300A, 0.4×30 cm; eluent 0-50%acetonitrile in 0.04 M triethylammonium buffer (pH 7.0); flow rate 1.5ml/min. The fractions containing LNA-oligos were pooled and evaporatedunder reduced pressure (oil pump and speed-vac system) during 12 h.

Hybridisation Data

Example 129

Thermostability of oligonucleotides containing monomers of formula V, X,Y and Z^(T), Z^(U), Z^(G), Z^(C), Z^(A), Z^(MeC). The thermostability ofthe LNA modified oligonucleotides were determined spectrophotometricallyusing a spectrophotometer equipped with a thermoregulated Peltierelement. Hybridisation mixtures of 1 ml were prepared containing eitherof 3 different buffers (10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl, 0.1 mM EDTA;10 mM Na₂HPO₄ pH 7.0, 0.1 mM EDTA; 3M tetramethylammoniumchlorid (TMAC),10 mM Na₂HPO₄, pH 7.0, 0.1 mM EDTA) and equimolar (1 μM or 1.5 μM)amounts of the different LNA modified oligonucleotides and theircomplementary or mismatched DNA or RNA oligonucleotides. Identicalhybridisation mixtures using the unmodified oligonucleotides wereprepared as references. The T_(m)'s were obtained as the firstderivative of the melting curves. Tables 1-4 summarise the results (LNAsare marked with bold). FIG. 2 illustrates the monomeric LNAs used. Thenomenclature V, X, Y and Z^(T), Z^(U), Z^(G), Z^(C), Z^(A), Z^(MeC)refer to structures V, X, Y and Z of FIG. 2. In the tables, thenucleobases of the LNA monomers are indicated. Furthermore, for the thioand amino variants of the LNA structure Z of the last two tables, thenomenclature used is, e.g., Z^(TS) and Z^(TNH), respectively.

LNAs containing structure Z were particularly thoroughly examined (seeTable 1). When three Z^(T) residues were incorporated into anoligonucleotide of mixed sequence the T_(m)'s obtained in NaCl bufferwith both complementary DNA (10) and RNA (16) oligonucleotides weresubstantially higher (RNA: roughly 7° C. and DNA: roughly 5° C. permodification) than the T_(m) of the corresponding duplexes withunmodified oligonucleotides (1 and 8). Similar results were obtainedwith LNAs containing two Z^(T) residues and either one Z^(G) (21 and24B) or Z^(U) (25), Z^(C) (69), Z^(MeC) (65), and Z^(A) (58) residues.When mismatches were introduced into the target RNA or DNAoligonucleotides the T_(m) of the LNA modified oligonucleotides in allcases dropped significantly (11-15A and 17; 18-20 and 22-24A; 26-31; 57and 59-60; 63-64 and 66, and 67), unambiguously demonstrating that theLNA modified oligonucleotides hybridise to their target sequencesobeying the Watson-Crick hydrogen bonding rules. In all cases the dropin T_(m) of the LNA modified oligonucleotides upon introduction ofmismatches was equal to or greater than that of the correspondingunmodified oligonucleotides (2-7 and 9; 33-38), showing that the LNAmodified oligonucleotide are at least as specific as their naturalcounterparts. A lowering of the ionic strength of the hybridisationbuffer (from 10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl, 0.1 mM EDTA to 10 mMNa₂HPO₄ pH 7.0, 0.1 mM EDTA) lowers the T_(m) of the LNA modifiedoligonucleotides for their complementary DNA oligos (40,41) or RNAoligonucleotides (40A, 41A). A similar effect is observed with theunmodified oligonucleotides and its complementary DNA oligo (39) or RNAoligo (39A).

Addition of 3M tetramethylammoniumchlorid (TMAC) to the hybridisationbuffer significantly increases the T_(m) of the LNA modifiedoligonucleotide for their complementary DNA oligos (10,21,25). Moreover,TMAC levels out the diffeneces in the T_(m)'s of the differentoligonucleotides which is observed in the NaCl buffer (lowest T_(m) inthe NaCl buffer 44° C. and highest 49° C. as opposed to 56° C. and 57°C. in TMAC). Introduction of mismatches substantially decreases theT_(m) of the LNA modified oligonucleotides for their DNA targets (11-13,18-20, and 26-28). A similar picture emerges with the unmodifiedreference oligonucleotides (1-4 and 32-35)

The data with the low salt buffer shows that LNA modifiedoligonucleotides exhibit a sensitivity to the ionic strength of thehybridisation buffer similar to normal oligonucleotides. From the T_(m)data with the TMAC buffer we infer that TMAC exhibits a T_(m) equalisingeffect on LNA modified oligonucleotides similar to the effect observedwith normal DNA oligonucleotides. LNA modified oligonucleotides retaintheir exquisite specificity in both hybridisation buffers.

The fully modified LNA oligonucleotide containing all four monomers (71and 75), the almost fully modified LNA oligonucleotide (except for a3′-terminal DNA nucleoside) containing both Z^(G) and Z^(T) (41 and 41A)and the partly modified oligonucleotide containing a central block ofZ^(T) and Z^(G) (40 and 40A) also exhibit substantially increasedaffinity compared to the unmodified control oligonucleotide (39 and 39A;1 and 8). This shows that LNAs of formula Z are very useful in theproduction of both fully and partly modified oligomers. We note that thealmost fully modified oligomer (41 and 41A) exhibits an unprecedentedhigh affinity for both complementory RNA (>93° C.) and DNA (83° C.). Asimilar extreme affinity (for both RNA and DNA) was observed with thealmost fully modified LNA oligomer containing exclusively Z^(T) (Table1: 52 and 53) and the fully modified LNA oligomer (71 and 75). Theaffinity of the partly modified poly-T oligonucleotide depended on thepositions and the number of Z^(T) monomers incorporated (44-51). Whereasthe T_(m)'s with RNA targets (45, 47, 49 and 51) in all cases werehigher than the corresponding unmodified oligonucleotides (43) one gavea lower T_(m) with the DNA target (46). Since mixed sequenceoligonucleotide containing 3 Z^(T) residues exhibited a substantiallyincreased affinity for their DNA (10) and RNA target (16) compared tothe unmodified reference oligonucleotides (1 and 8) this suggests thatother binding motifs than Watson-Crick (such as for example theHoogsteen binding motif) are open to poly-T oligonucleotides and thatthese binding motifs are somewhat sensitive to the precise architectureof the modified oligonucleotide. In all cases introduction of singlebase mismatches into the complex between the fully Z^(T) modified poly-Toligonucleotide and a DNA target (54-56) resulted in a significant dropin T_(m).

Oligonucleotides containing either LNAs of structures V (Table 2), X(Table 3) and Y (Table 4) were analysed in the context of fully andpartly modified poly-T sequences. The fully modified oligonucleotides ofstructure V and Y exhibited an increase in T_(m) (albeit much lower thanthe Z^(T) modified oligonucleotides) with both RNA (Table 2, 14 andTable 4, 14) and DNA targets (Table 2, 13, and Table 4, 13) compared tothe unmodified oligonucleotides (Table 1, 42 and 43). The partlymodified oligonucleotides containing monomers of structure V and Ybehaved similarly to partly modified oligonucleotides containing Z^(T)and probably this is due to the homopolymer nature of the sequence asoutlined above. Oligonucleotides containing X^(T) in all cases exhibiteda much reduced T_(m) compared to the reference DNA oligonucleotides.

Example 130

A fully modified LNA oligonucleotide form stable hybrids withcomplementary DNA in both the anti-parallel and the parallelorientation. A full modified LNA oligonucleotide was hybridised to itscomplementary DNA in both the anti-parallel and the parallelorientation. Hybridisation solutions (1 mL) contained 10 mM Na₂HPO₄ (pH7), 100 mM NaCl and 0.1 mM EDTA and 1 μM of each of the twoolignucleotides. As shown in Table 1 both the anti-parallel (71) and theparallel binding orientation (77) produces stable duplexes. Theanti-parallel is clearly the most stable of the two. However, even theparallel duplex is significantly more stable than the correspondinganti-parallel duplex of the unmodified DNA oligonucleotides (Table 1,1).

Example 131

LNA monomers can be used to increase the affinity of RNA oligomers fortheir complementary nucleic acids. The thermostability of complexesbetween a 9-mer RNA oligonucleotide containing 3 LNA-T monomers (Z^(T))and the complementary DNA or RNA oligonucleotides were measuredspectrophotometrically. Hybridisation solutions (1 ml) containing 10 mMNa₂HPO₄, pH 7.0, 100 mM NaCl, 0.1 mM EDTA and 1 μM of each of the twooligonucleotides. Identical hybridisation mixtures using the unmodifiedRNA oligonucleotides were measured as references. As shown in Table 5the LNA modified RNA oligonucleotide hybridises to both itscomplementary DNA (1) and RNA (3) oligonucleotide. As previouslyobserved for LNA modified DNA oligonucleotides, the binding affinity ofthe LNA modified RNA oligonucleotide is strongest to the RNA complement(3). In both cases the affinity of the LNA modified RNA oligonucleotideis substantially higher than that of the unmodified controls (2 and 4).Table 5 also shows that the specificity towards both DNA and RNA targetsare retained in LNA modified RNA oligonucleotides.

Example 132

LNA-LNA base pairing. RNA or DNA oligonucleotides containing three Z^(T)LNA monomers or an oligonucleotide composed entirely of LNA Z monomerswere hybridised to complementary unmodified DNA oligonucleotides or DNAoligonucleotides containing three Z^(A) LNA monomers and the T_(m) ofthe hybrids were measured spectrophotometrically. Hybridisationsolutions (1 ml) contained 10 mM Na₂HPO₄, pH 7.0, 100 mM NaCl and 0.1 mMEDTA and 1 μM of each of the two oligonucleotides. As shown in Table 6all the LNA modified oligonucleotides hybridises to the complementary,unmodified DNA oligonucleotides (2 and 3) as well as the complementaryLNA modified oligonucleotides (4, 5 and 6). As observed previously thepresence of LNA monomers in one strand of a hybrid (2 and 3) increasesthe T_(M) significantly compared to the unmodified control hybrid (1).The presence of LNA-LNA base pairs in the hybrid increases the T_(M)even further (4 and 5) Moreover, a highly stable hybrid can be formedbetween a fully modified LNA oligonucleotide and a partly LNA-Z^(A)modified DNA oligonucleotide (6). This constitutes the first example ofLNA-LNA base pairs in a hybrid.

Example 133

An LNA all-phosphoromonothioate oligonucleotide display relatively lessdecreased thermostability towards complementary DNA and RNA than thecorresponsing all-phosphorothioate DNA oligonucleotide. Thethermostability of an all-phosphoromonothioate DNA oligonucleotidecontaining three Z^(T) LNA monomers (LNA oligonucleotide) and thecorresponding all-phosphoromonothioate reference DNA oligonucleotidetowards complementary DNA and RNA was evaluated under the sameconditions as described in Example 132, however without EDTA (Table 7).It was observed that the LNA all-phosphoromonothioate oligonucleotidecontaining three LNA Z^(T) monomers displayed only weakly decreasedthermostability (Table 7, 3 and 4) when compared to the correspondingreference LNA oligonucleotide (Table 1, 10 and 16). The correspondingall-phosphoromonothioate DNA oligonucleotide (Table 7, 1 and 2)displayed significantly decreased thermostability when compared to thecorresponding reference DNA oligonucleotide (Table 1, 1 and 8). This hasimportant possible implications on the use of all- or partiallyphosphoromonothioate LNA oligonucleotides in antisense and othertherapeutic applications. Thus, the compatibility of LNA monomers andunmodified monomers in an phosphoromonothioate oligonucleotide has beendemonstrated. It can be anticipated that such constructs will displayboth Rnase H activity and nuclease resistance in addition to the LNAenhanced hybridisation characteristics.

Example 134

2′-Thio-LNA display nucleic acid recognition properties comparable withthose of LNA (Monomer Z). The hybridisation conditions were as describedin Example 132, however without EDTA. The results for the 2′-thio-LNAs(Table 8) clearly indicate a positive effect on the thermal stability ofduplexes towards both DNA and RNA by the introduction of 2′-thio-LNAmonomer U^(S) (The monomers correspond to formula Z of FIG. 2 where themethyleneoxy bridge has been substituted with a methylenethio bridge).This effect (ΔT_(m)˜+5° C./modification towards DNA; ΔT_(m)˜+8°C./modification towards RNA) is comparable with that observed for parentLNA. The picture is complicated by the simultaneous introduction of twomodifications (the 2′-thio functionality and uracil instead of thymine).However, as we have earlier observed identical melting temperatures forthe LNA thymine and uracil monomers, and as the references containing2′-deoxyuridine instead of thymidine, if anything, would be expected todisplay lower T_(m) values, the comparison is relevant.

Example 135

2′-Amino-LNA (Monomer Z^(TNH)) and 2′-Methylamino-LNA (Monomer Z^(TNMe))display nucleic acid recognition properties comparable with those ofparent LNA (Monomer Z). The hybridisation conditions were as describedin Example 132, however without EDTA. The melting results for the2′-amino-LNAs (Table 9) clearly indicate a positive effect on thethermal stability of duplexes towards DNA and RNA by introduction ofeither 2′-amino-LNA monomers T^(NH) or T^(NMe) (The monomers correspondto formula Z of FIG. 2 where the methyleneoxy bridge has beensubstituted with a methyleneamino bridge or methylene-(N-methyl)aminobridge, respectively). This effect (ΔT_(m)˜+3° C./modification towardsDNA and ΔT_(m)˜+6 to +8° C./modification towards RNA) is comparable tothat of parent LNA. It is notheworthy, that the increased thermalaffinity is also observed with an oligo composed of a mixture of2′-alkylamino-LNA monomers and nonalkylated 2′-amino-LNA monomers.

LNA and LNA modified oligonucleotides as a substrates for enzymes

Example 136

3′-Exonucleolytic stability of oligomers 5′-V^(T) ₁₃T and 5′-Z^(T) ₁₃T.A solution of oligonucleotides (0.20D) in 2 ml of the following buffer(0.1 M Tris-HCl, pH 8.6, 0.1 M NaCl, 14 mM MgCl₂) was digested at 25° C.with 1.2 U SVPDE (snake venom phosphodiesterase). During digestion, theincrease in absorbance at 260 nm was followed. Whereas the unmodifiedcontrol T₁₄ was fully degraded after 10 min of degradation, 5-Z^(T) ₁₃Tand 5′-V^(T) ₁₃T remained intact for 60 min.

Example 137

LNA modified oligos as substrates for T4 polynucleotide kinase. 20pmoles of each primer (FP2: 5′-GGTGGTTTGTTTG-3′; DNA probe), (AL2:5′-GGTGGTTTGTTTG-3′, LNA nucleosides in bold) and (AL3:5′-GGTGGTTTGTTTG-3′, LNA nucleosides in bold) was mixed with T4polynucleotide Kinase (5 Units; New England Biolabs) and 6 μl γ-³²PATP(3000 Ci/mmol, Amersham) in a buffer containing 70 mM Tris-HCl (pH 7.6),10 mM MgCl₂, 5 mM dithiotretiol (final volume 20 μA. The samples wereincubated 40 min at 37° C. and afterwards heated to 65° C. for 5 min. Toeach of the reactions were added 2 μl of tRNA (1 μg/μl), 29 μl of a 3Mammonium acetate and 100 μl of ethanol. The reactions were incubated at−20° C. for 30 min. and the labelled oligos were precipitated bycentrifugation at 15000 g for 30 min. The pellet was resuspended in 20μl H₂O. The samples (1 μA were mixed with a loading buffer (formamide(pH 8.0), 0.1% xylene cyanol FF, 0.1% bromophenol blue and 10 mM EDTA)and electrophoresed on a denaturing polyacrylamide gel (16%acrylamide/bisacrylamide solution, 7 M urea, 1×TBE and 0.1 mM EDTA) in aTBE running buffer (90 mM Tris-HCl (pH 8.3), 90 mM boric acid and 2.5 mMdisodium EDTA-2H₂O). The gel was dried on a gel dryer (BioRad model 583)and autoradiographed to a X-ray film (CL-XPosure film, Pierce 34075) for20 min. The result is shown in FIG. 6 (FP2: lane 1 and 2; AL2: lane 3and 4; AL3: lane 5 and 6). Three conclusions can be drawn on the basisof this experiment. Firstly, it can be concluded that partly and fullyLNA modified oligos are excellent mimics of natural nucleic acid intheir ability to act as substrate for a nucleic acid specific enzymelike polynucleotide kinase. Secondly, it can be concluded that LNAmodified oligos can be efficiently precipitated by procedures normallyemployed to precipitate standard nucleic acids. In fact, the relativesignal intencities of the unmodified (lane 1,2), partly (lane 3,4) andfully modified oligos (lane 5,6) in the autoradiogram suggests that themore LNA nucleosides a standard DNA oligo contains the more efficientlyit can be precipitated by salt/alcohol procedures. Thirdly, the similarpositions of the signal in the autoradiogram of the unmodified, partlyand fully modified oligos shows that incorporation of LNA nucleosidesinto a DNA oligo does not alter its electrophoretic mobility inpolyacrylamide gels.

Example 138

3′-End labelling of LNA-containing oligonucleotides with terminaldeoxynucleotidyl transferase. Oligonucleotides containing LNA monomerswere 3′ end-labelled using the enzyme terminal deoxynucleotidyltransferase. The sequence and extent of LNA modification were as follows(where LNA monomers are in bold):

Control 5′ GGT GGT TTG TTT G 3′ (1) 5′ GGT GGT TTG TTT G 3′ (2) 5′GGT GGT TTG TTT G 3′ (3) 5′ GGT GGT TTG TTT G 3′

Oligonucleotide (50 μmol) was incubated with 250 μCi [α-³²P]ddATP (3000Ci/mmol) and 100 Units terminal deoxynucleotidyl transferase in 250 μl100 mM cacodylate buffer pH 7.2, 2 mM CoCl₂ and 0.2 mM 2-mercaptoethanolat 37° C. for 2 hours. The reaction was then stopped by adding formamideloading buffer and heating to 100° C. for 5 min before placing on ice.Samples (0.2 μmol) were run on a 19% acrylamide gel containing 7M ureaand the percentage incorporation of radioactivity into theoligonucleotide bands was quantified by means of a phosphorimager(Molecular Dynamics). The results show incorporation of radioactivity inall cases, including the oligonucleotide with a high LNA content:Control 94.9%, (1) 39.7%, (2) 83.7%, (3) 31.7%. We conclude that LNAmodified oligos are substrates for the TdT enzyme.

Example 139

The ability of terminal deoxynucleotidyl transferase (TdT) to tail LNAmodified oligonucleotides depends on the design of the oligomer. Thefollowing 15mer primers and a mixture of 8 to 32 base oligonucleotidemarkers were 5′ end labelled with [γ³³P] ATP and T4 polynucleotidekinase (where LNA monomers are in bold):

P1 5′-TGC ATG TGC TGG AGA-3′ P2 5′-GC ATG TGC TGG AGA T-3′ PZ15′-TGC ATG TGC TGG AGA-3′ PZ2 5′-GC ATG TGC TGG AGA T-3′Reactions were boiled for 5 min after labelling to remove any PNKactivity. Four picomoles of each labelled primer, 25 U terminaldeoxynucleotidyl transferase and 16 μM dATP were incubated in 25 μl 100mM cacodylate buffer pH 7.2, 2 mM CoCl₂ and 0.2 mM 2-mercaptoethanol for90 min at 37° C. The reactions were stopped by the addition of formamidestop solution and the reaction products run on a 19% polyacrylamide 7 Murea gel with the labelled markers. Autoradiography using Biomax filmwas carried out on the dry gel. As shown in FIG. 22, P1 (lane 2), P2(lane 4) and PZ1 (lane 3) all gave a tail estimated at greater than 70bases long on the basis of the 8-32 base marker (lanes 1 and 6). PrimerPZ2 (lane 5) was not extended under these reaction conditions. Weconclude that the TdT enzyme will tolerate LNA monomers within theoligonucleotide, but not at the extreme 3′ end.

Example 140

LNA-thymidine-5′-triphosphate (LNA-TTP) as a substrate for terminaldeoxynucleotidyl transferase (TdT). In order to test the ability of thetriphosphate of LNA-TTP (Example 123) to be accepted by terminaldeoxynucleotidyl transferase as a substrate, an oligonucleotide tailingreaction was performed. A 15mer primer (sequence: 5′-TGC ATG TGC TGGAGA-3′) and a mixture of 8 to 32 base oligonucleotide markers were 5′end labelled with [γ³³P] ATP and T4 polynucleotide kinase. Reactionswere boiled for 5 min after labelling to remove any PNK activity. Fourpicomoles of the labelled primer, 25 U terminal deoxynucleotidyltransferase and 32, 64 or 128 μM dTTP or LNA-TTP were incubated in 25 μl100 mM cacodylate buffer pH 7.2, 2 mM CoCl₂ and 0.2 mM 2-mercaptoethanolfor 90 min at 37° C. The reactions were stopped by the addition offormamide stop solution and the reaction products run on a 19%polyacrylamide 7M urea gel with the labelled markers. Autoradiographyusing Biomax film was carried out on the dry gel. As shown in FIG. 10,reactions with either 32 μM dTTP (lane B), 64 μM dTTP (lane C) or 128 μMdTTP (lane D) all produced tailed oligonucleotides which on the basis onthe 8-32 oligonucleotide marker (outermost left and rigth lanes) wereestimated at greater than 100 nucleotides. The LNA-TTP reactions (32 μMdTTP (lane E), 64 μM dTTP (lane F) or 128 μM dTTP (lane G)) all resultedin the primer being extended by one base and ˜50% of this being extendedby a further base. This result is very similar to that obtained withribonucleotides and TdT. We conclude that LNA derived triphosphates canbe recognised and incorporated into a DNA oligonucleotide by the TdTenzyme. This latter finding that LNA-TTP can bind to the polymeraseunderscores the possibility of successfully using LNA-monomerderivatives as nucleoside drugs.

Example 141

Exonuclease free Klenow fragment DNA polymerase I can incorporate LNAAdenosine, Cytosine, Guanosine and Uridine-5′-triphosphates (LNA ATP,LNA CTP, LNA GTP, LNA UTP) into a DNA strand. A primer extension assaywas used to evaluate the LNA NTP's (see Example 123), ribonucleotides,as substrates for exonuclease free Klenow fragment DNA polymerase I(EFK). The assay used a ³³P 5′ end labelled 15mer primer hybridised toone of four different 24mer templates. The sequences of the primer andtemplates are (LNA monomer in bold):

Primer 5′ TGCATGTGCTGGAGA 3′ Template 1 3′ ACGTACACGACCTCTACCTTGCTA 5′Template 2 3′ ACGTACACGACCTCTCTTGATCAG 5′ Template 3 3′ACGTACACGACCTCTTGGCTAGTC 5′ Template 4 3′ ACGTACACGACCTCTGAACTAGTC 5′

One picomole ³³P labelled primer was hybridised to 2 picomoles oftemplate in ×2 Klenow buffer. To this was added either 4 μM dNTPαS or500 μM LNA NTP or a mixture of 4 μM dNTPαS and 500 μM LNA NTP. Two unitsof EFK DNA polymerase was added to each reaction. 2mU inorganicpyrophosphatase was added to each of the reactions. Primer plus templateplus enzyme controls were also carried out. All reactions were carriedout in a total volume of 20 μl. The reactions were incubated at 37° C.for 3 min. Reactions were then stopped by the addition of 10 μlformamide EDTA stop solution. Reaction products were separated on a 19%polyacrylamide 7M urea gel and the product fragments sized by comparisonwith a ³³P labelled 8 to 32 base oligonucleotide ladder after exposureto Kodak Biomax autoradiography film.

FIG. 20 shows the result with LNA-UTP using template 1. The tracks(1-12) correspond to the following reactions: Incorporation of LNA UTPby EFK. Lane 1-Primer, template and enzyme. Lane 2-plus dTTPαS. Lane3-plus LNA UTP. Lane 4-plus dTTPαS and dGTPαS. Lane 5-plus LNA UTP anddGTPαS. Lane 6-plus dATPαS, dGTPαS and dTTPαS. Lane 7-plus LNA UTP,dCTPαS, dGTPαS and dTTPαS. Lane 8-plus dGTPαS. Lane 9-plus dCTPαS,dGTPαS and dTTPαS. Lane 10-plus LNA UTP, dATPαS, dCTPαS and dGTPαS. Lane11-plus dATPαS, dCTPαS and dGTPαS. Lane 12-all 4 dNTPαS. The laneseither side show the 8-32 base oligonucleotide markers used for sizingthe products.

As is evident from FIG. 20, LNA UTP is specifically incorporated as a“T”. Further extension from an LNA UTP terminated 3′ end with dNTPαS isvery slow.

FIG. 21 shows the result with LNA-ATP, LNA CTP, and LNA GTP usingtemplate 2-4. The tracks (1-21) correspond to the following reactions:Lanes 1, 7, 13 and 17-primer, template and enzyme. Lane 2-plus dGTPαS.Lane 3-plus dATPαS and dGTPαS. Lane 4-plus LNA GTP. Lane 5-plus dGTPαSand LNA ATP. Lane 6-plus LNA ATP and LNA GTP. Lane 8-plus dATPαS. Lane9-plus dATPαS and dCTPαS. Lane 10-plus LNA ATP. Lane 11-plus dCTPαS andLNA ATP. Lane 12-plus dATPαS and LNA CTP. Lane 14-plus dTTPαS. Lane15-plus dGTPαS and dTTPαS. Lane 16-plus dTTPαS and LNA GTP. Lane 18-plusdCTPαS. Lane 19-plus dCTPαS and dTTPαS. Lane 20-plus LNA CTP. Lane21-dTTPαS and LNA CTP. The lanes either side show the 8-32 baseoligonucleotide markers used for sizing the products.

The experiments using template 2 (track 1-6), show that LNA GTP is ableto produce the +1 product with efficient extension of the primer (track4). The addition of dGTPαS and LNA ATP results in mainly the +2 product(track 5). This is from the incorporation of dGTPαS to give the +1product followed by extension with LNA ATP. There is evidence of a smallamount of +3 product from the consecutive incorporation of LNA ATP. Theexperiments using Template 3 (tracks 7-12) show that LNA ATP isefficiently incorporated to give the +1 product (track 10). Extension ofthis product with dCTPαS is slow (track 11). The addition of dATPαS andLNA CTP results in the +2 and +3 products (track 12). The absence of anysignificant +1 product shows that the addition of the first LNA CTP isefficient, but that the addition of the second LNA CTP is slow. Theresults from experiments on Templates 1 (tracks 13-16) and 4 (tracks17-21) show similar trends to those on the other templates. LNA CTP isefficiently incorporated to give the +1 product on Template 4 (track20). Extension of this product by dTTPαS is again slow (track 21). Theaddition of LNA GTP and dTTPαS to reactions on Template 1 results in the+2 product (track 16). Again this shows that the addition of a singleLNA triphosphate is quite efficient, but that the addition ofconsecutive LNA triphosphates is slow.

Example 142

LNA monomers can be used to enhance the resistance of an oligonucleotideto digestion by exonuclease III. In order to test the resistance of theLNA containing oligonucleotides to Exonuclease III degradation thefollowing reaction was performed. The following 15mer primers and 8 to32 base oligonucleotide markers were 5′ end labelled with [γ³³P] ATP andT4 polynucleotide kinase (LNA monomer in bold):

P2 5′-GC ATG TGC TGG AGA T-3′ PZ2 5′-GC ATG TGC TGG AGA T-3′Reactions were boiled for 5 min after labelling to remove any PNKactivity. 8 picomoles of each primer was hybridised to 25 pmolesTemplate (sequence: 3′-ACG TAC ACG ACC TCT ACC TTG CTA-5′) in ×2 Klenowbuffer. 10 Units of Exonuclease III was added to each of the reactions.Controls were also set up which had 1 μl water added in place of theenzyme. The reactions were incubated at 37° C. for 5 min. The reactionswere stopped by the addition of 10 μl formamide/EDTA stop solution. Thereactions were heated at 95° C. for 3 min before loading onto a 19%polyacrylamide 7M urea gel. The gel was fixed in 10% acetic acid/10%methanol before transferring to 3 MM paper and drying. The dried gel wasexposed to a phosphor screen for 3 hours. The phosphor screen wasanalysed on the Molecular Dynamics Storm 860 instrument using ImageQuantsoftware. The phosphor screen analysis showed that in the absence of theenzyme the P2 full length band was 99% of the signal and PZ2 full lengthband was 96% of the signal. In the presence of the enzyme only 20% ofthe P2 full length product was left after the 5 minute incubation.However, 62% of the full length PZ2 product remained after the sametreatment. This shows that a single LNA monomer at the 3′ end of anoligonucleotide can enhance the resistance to degradation by exonucleaseIII.PCR Applications

Example 143

LNA monomers can be used to significantly increase the performance ofbiotinylated-DNA oligos in the sequence specific capture of PCRamplicons in a MTP format. Two DIG labelled amplicons from pUC19 weregenerated by PCR amplification as follows:

PCR Reaction Mixture for Amplicon 1

1 μl pUC19 (1 ng/μl),

1 μl reverse primer (5′-AACAGCTATGACCATG-3′) (20 μM),

1 μl forward primer (5′-GTAAAACGACGGCCAGT-3′) (20 μM),

10 μl dUTP-mix (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 6 mM dUTP),

1.5 μl DIG-11-dUTP (1 mM)

10 μl 10×Taq buffer (Boehringer Mannheim incl MgCl₂)

1 μl Taq polymerase (Boehringer Mannheim) 5 U/μl

H₂O ad 100 μl

PCR Reaction Mixture for Amplicon 2

1 μl pUC19 (1 ng/μl),

0.4 μl primer 3 (5′-GATAGGTGCCTCACTGAT-3′) (50 μM),

0.4 μl primer 4 (5′-GTCGTTCGCTCCAAGCTG-3′) (50 μM),

10 μl dUTP-mix (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 6 mM dUTP),

1.5 μl DIG-11-dUTP (1 mM)

10 μl 10× Taq buffer (Boehringer Mannheim incl MgCl₂)

1 μl Taq polymerase (Boehringer Mannheim) 5 U/μ1

H₂O ad 100 μl

PCR reaction: (Cycler: Perkin Elmer 9600) 94° C. 5 min; add polymerase;94° C. 1 min, 45° C. 1 min, 70° C. 2 min (29 cycles) 72° C. 10 min.

10 μl from each PCR reaction was analysed on a standard agarose gel andthe expected fragments of approximately 100 by and 500 by were observed.

10 μl of DIG-labelled amplicon 1 or amplicon 2 was mixed with 5 μmol of5′ biotinylated capture probe in 1×SSC (0.15 M NaCl, 15 mM citrate, pH7.0) in a total volume of 450 μl. The following capture probes wereused: B-DNA1 (biotin-ATGCCTGCAGGTCGAC-3′; DNA probe specific foramplicon 1), B-DNA2 (biotin-GGTGGTTTGTTTG-3′; DNA probe specific foramplicon 2) and B-LNA2 (biotin-GGTGGTTTGTTTG-3′, LNA nucleosides inbold; LNA probe specific for amplicon 2). Reactions were heated to 95°C. for 5 min in order to denature amplicons and allowed to cool at 25°C. for 15 min to facilitate hybridisation between the probe and thetarget amplicon strand. After hybridisation 190 μl of each reaction weretransferred to a streptavidin coated micro plate (Pierce, cat. no.15124) and incubated for one hour at 37° C. After washing the plate withphosphate buffered saline (PBST, 0.15 M Na⁺, pH 7.2, 0.05% Tween 20,3×3000), 200 μl of peroxidase labelled anti-DIG antibodies were added(Boehringer Mannheim, diluted 1:1000 in PBST). Plates were incubated for30 min at 37° C. and washed (PBST, 3×3000). Wells were assayed forperoxidase activity by adding 100 μl of substrate solution (0.1 Mcitrate-phosphate buffer pH 5.0, 0.66 mg/ml ortho-pheylenediaminedihydrochloride, 0.012% H₂O₂). The reaction was stopped after 8 min byadding 100 μl H₂SO₄ (0.5 M) and the absorbance at 492 nm was read in amicro plate reader. As shown in FIG. 3, the unmodified bio-DNAs captureprobes (B-DNA1 and B-DNA2) both behave as expected, i.e. they eachcapture only their target PCR amplicon. Compared to the B-DNA1 probe theB-DNA2 probe is rather inefficient in capturing its cognate amplicon.The capture efficiency of the B-DNA2 probe, however, can be dramaticallyimproved by substituting 12 of its 13 DNA nucleosides by thecorresponding LNA nucleosides. As shown in FIG. 3 the use of the B-LNA2probe in place of the B-DNA2 probe leads to a more that 10 fold increasein the sensitivity of the assay. At the same time the B-LNA2 retains theability of the un-modified B-DNA2 to efficiently discriminate betweenthe related and non-related amplicon, underscoring the excellentspecificity of LNA-oligos. We conclude that 1) biotin covalentlyattached to an LNA modified oligo retains its ability to bind tostreptavidin, 2) that LNA modified oligos works efficiently in a MTPbased amplicon capture assay and that 3) LNA offers a means todramatically improve the performance of standard DNA oligos in theaffinity capture of PCR amplicons.

Example 144

An LNA substituted oligo is able to capture its cognate PCR amplicon bystrand invasion. Two identical sets of 10 μl reactions of amplicon 1 or2 (prepared as in Example 143) were mixed with either 1, 5 or 25 μmol ofthe B-LNA2 capture probe (biotin-GGTGGTTTGTTTG-3′, LNA nucleosides inbold; probe specific for amplicon 2) in 1×SSC (0.15 M NaCl, 15 mMcitrate, pH 7.0) in a total volume of 450 μl. One set of reactions wereheated to 95° C. for 5 min in order to denature amplicons and allowed tocool to 25° C. to facilitate hybridisation between the probe and thetarget amplicon strand. The other set of reactions were left withoutdenaturation. From each of the reactions 190 μl were transferred to astreptavidin coated micro plate (Pierce, cat. no. 15124) and incubatedfor one hour at 37° C. After washing the plate with phosphate bufferedsaline (PBST, 0.15 M Na⁺, pH 7.2, 0.05% Tween 20, 3×3000), 200 μl ofperoxidase labelled anti-DIG antibodies were added (Boehringer Manheim,diluted 1:1000 in PBST). Plates were incubated for 30 min at 37° C. andwashed (PBST, 3×3000). Wells were assayed for peroxidase activity byadding 100 vl of substrate solution (0.1 M citrate-phosphate buffer pH5.0, 0.66 mg/ml ortho-pheylenediamine dihydrochloride, 0.012% H₂O₂). Thereaction was stopped after 10 min by adding 100 μl H₂SO₄ (0.5 M) and theabsorbance at 492 nm was read in a micro plate reader. When ampliconsare denaturated prior to hybridisation with the capture probe (FIG. 4A)we observe an efficient and sequence specific amplicon capture similarto that shown in Example 143. Increasing the concentration of the B-LNA2from 1 to 5 μmol leads to an increase in capture efficiency. A furtherincrease to 25 μmol of probe results in a decreased signal. Thisobservation is consistent with saturation of the available biotinbinding sites on the streptavidin MTP. When amplicons are notdenaturated prior to hybridisation with the capture probe (FIG. 4B) wealso observe an efficient and sequence specific amplicon capture. Infact, the data shows that amplicon capture without denaturation are aseffective and specific as amplicon capture with denaturation. Thisstrongly indicates that the Bio-LNA2 probe is capable of binding to itstarget sequence by strand invasion. To our knowledge, this constitutesthe first example ever of sequence specific targeting of dsDNA underphysiological salt conditions by a mixed purine/pyrimidine probe. Asidefrom its potential to significantly simplify a range of basic researchand DNA diagnostic procedures this unexpected property of LNA modifiedoligos can be foreseen to be of major importance in the development ofefficient new drugs by the antisense, and in particular anti-geneapproach.

Example 145

An LNA substituted oligo, immobilised on a solid surface functionefficiently in the sequence specific capture of a PCR amplicon. Wells ofa streptavidin coated micro-titer plate (Boehringer Mannheim) wereincubated for 1 hour with either 5 μmol of the B-DNA2 probe(biotin-GGTGGTTTGTTTG-3′; DNA probe specific for amplicon 2) or theB-LNA2 probe (biotin-GGTGGTTTGTTTG-3′, LNA nucleosides in bold; LNAprobe specific for amplicon 2) in a total volume of 100 μl 1×SSC (0.15 MNaCl, 15 mM citrate, pH 7.0). In total, four wells were incubated withthe B-DNA2 probe, four wells with the B-LNA2 probe and four wells wereincubated with buffer alone. After incubation the wells were washedthree times with 1×SSC. DIG-labelled amplicon 1 (600 or amplicon 2 (60μl) (prepared as in Example 143) were mixed with 540 μl of 1×SSC, heatdenaturated at 95° C. for 5 min., and transferred (1000 to the microplate wells. Two of the wells containing either B-DNA2, B-LNA2 or nocapture probe received amplicon1 and two of the wells containing B-DNA2,B-LNA2 or no capture probe received amplicon2. After 1 hour at 37° C.the plate was washed 3 times with phosphate buffered saline (PBST, 0.15M Na⁺, pH 7.2, 0.05% Tween 20, 3×300 μl) and 200 μl of peroxidaselabelled anti-DIG antibodies were added (Boehringer Mannheim, diluted1:1000 in PBST). Plates were incubated for 30 min at 37° C. and washed 3times with 300 μl PBST. Wells were assayed for peroxidase activity byadding 100 μl of substrate solution (0.1 M citrate-phosphate buffer pH5.0, 0.66 mg/ml ortho-pheylenediamine dihydrochloride, 0.012% H₂O₂). Thereaction was stopped after 6 min by adding 100 μl H₂SO₄ (0.5 M) and theabsorbance at 492 nm was read in a micro plate reader. As shown in FIG.5, the LNA modified capture probe (B-LNA2) captures its specificamplicon (amplicon2) very efficiently and significantly better (approx.five fold increase in sensitivity) than the corresponding unmodified DNAcapture probe (B-DNA2). No signal is obtained when the B-LNA2 probe isincubated with the unrelated amplicon (amplicon1) underscoring theexquisite specificity of the B-LNA2 probe. We conclude that LNA modifiedoligos function efficiently in the sequence specific capture of PCRamplicons when immobilised on a solid surface. We further conclude thatthe use of LNA modified oligos in place of standard DNA oligos providefor a better signal to noise ratio. Thus, LNA offers a means tosignificantly improve the performance of current DNA based assays thatutilises immobilised capture probes, like for instance the array formatwherein multiple immobilised probes are used to simultaneously detectthe occurrence of several different target sequences in a sample.

Example 146

Fully mixed LNA monomers can be used to significantly increase theperformance of immobilised biotinylated-DNA oligos in the sequencespecific capture of PCR amplicons in a MTP format. Three DIG labelledamplicons from Nras sequence (ref.: Nucleic Acid Research, 1985, Vol.13, No. 14, p 52-55) were generated by PCR amplification as follows:

PCR primers: Forward primer: 5′-CCAGCTCTCAGTAGTTTAGTACA-3′bases 701-723 according to the NAR reference. 910 bp reverse primer:5′-GTAGAGCTTTCTGGTATGACACA-3′bases 1612-1590 (reverse sequence according to NAR ref.).600 bp reverse primer: 5′-TAAGTCACAGACGTATCTCAGAC-3′bases 1331-1308 (reverse sequence according to NAR ref.).200 bp reverse primer: 5′-CTCTGTTTCAGACATGAACTGCT-3′bases 909-886 (reverse sequence according to NAR ref.).

PCR reaction mixture for Nras amplicons: 2.3 μl human placental genomicDNA (440 ng/μl), 50 μl 10×PCR buffer (without MgCl₂ Perkin Elmer), 30 μl25 mM MgCl₂, 50 μl dNTP-mix (2 mM dATP, dCTP, dGTP and 1.8 mM dTTP), 10μl 1 mM Dig-11-dUTP, 10 μl 25 μM forward primer, 10 μl 25 μM reverseprimer, 5 μl 5 U/μl AmpliTaq Gold (Perkin Elmer) and water ad 500 μl.PCR reaction: The above mixture was made for all the Nras PCR products.The only difference being reverse primer 910 bp, 600 by or 200 by addedonce at a time. The PCR mixtures were aliquoted into ten PCR tubes eachand cycled in a Perkin Elmer 9600 at the following conditions: 95° C. 3min; 55° C. 2 min, 72° C. 3 min, 95° C. 1 min (30 cycles); 55° C. 2 min,72° C. 10 min and 4° C. soak. 10 μl from each PCR reaction was analysedon a standard agarose gel and the expected fragments of approximately910 bp, 600 by and 200 by were observed. Assay conditions: Wells of astreptavidin coated micro-titer plate (Boehringer Mannheim; bindingcapacity of 20 μmol biotin per well) were incubated for 1 hour in 5×SSCT(0.75 M NaCl, 75 mM citrate, pH 7.0, 0.1% Tween 20) at 37° C. witheither 1 μmol of DNA Nras Cap A (biotin-5′-TTCCACAGCACAA-3′), LNA/DNANras Cap A (biotin-5′-TTCCACAGCACAA-3′), LNA Nras Cap A(biotin-5′-TTCCACAGCACAA-3′), DNA Nras Cap B(biotin-5′-AGAGCCGATAACA-3′), LNA/DNA Nras Cap B(biotin-5′-AGAGCCGATAACA-3′) or LNA Nras Cap B(biotin-5′-AGAGCCGATAACA-3′); LNA nucleosides in bold. The Nras Cap Acapture probes capture amplicons Nras 910, Nras 600 and Nras 200. NrasCap B capture probes capture specific amplicons Nras 910 and Nras 600.After incubation with the different capture probes, the wells werewashed in 5×SSCT and 5 μl native or denatured (95° C. 5 min and 10 minon ice) DIG-labelled amplicons (Nras 910, Nras 600 or Nras 200) in 95 μl1×SSCT (0.15 M NaCl, 15 mM citrate, pH 7.0, 0.1% Tween 20) were addedper well and incubated for 1 hour at 37° C. The wells were washed threetimes in phosphate buffered saline (1×PBST, 0.15 M Na⁺, pH 7.2, 0.05%Tween 20) and incubated 30 min at 37° C. with 200 μl peroxidase labelledanti-DIG antibodies (Boehringer Mannheim, diluted 1:1000 in 1×PBST).Finally the wells were washed three times in 1×PBST and assayed forperoxidase activity by adding 100 μl of substrate solution (0.1 Mcitrate-phosphate buffer pH 5.0, 0.66 mg/ml ortho-pheylenediaminedihydrochloride, 0.012% H₂O₂) the reaction was stopped after 9 min byadding 100 μl 0.5 M H₂SO₄ and diluted 4 times in H₂SO₄ before theabsorbance at 492 nm was read in a micro-titer plate reader. As shown inFIG. 23A, capture probes spiked with 12 LNA nucleosides (LNA Nras Cap Aand LNA Cap B) capture very efficiently the specific amplicons withoutprior denaturation (native amplicons). Capture probes spiked with 4 LNAnucleosides (LNA/DNA Nras Cap A and LNA/DNA Nras Cap B) capture the sameamplicons with a lower efficiency and the DNA capture probes (DNA NrasCap A and DNA Nras Cap B) do not capture the specific amplicons at all.The control amplicon, Nras 200, are not captured by the LNA Cap B or theLNA/DNA Nras Cap B probes demonstrating the exquisite specificity of theLNA spiked capture probes. FIG. 23B shows the same experiment performedwith denatured amplicons. Essentially the same picture emerges with theessential difference that capture efficiencies are generally increased.We conclude that LNA modified oligos containing mixed LNA nucleosides(A, T, G or C LNA nucleosides) function efficiently in sequence specificcapture of PCR amplicons when immobilised on a solid surface. We furtherconclude that LNA offers a means to construct capture probes that willfunction efficiently in amplicon capture without prior denaturation i.e.capture by strand displacement. This ability facilitates a significantsimplification of current amplicon detection formats based on DNA.

Example 147

LNA modified oligos function as primers for nucleic acid polymerases.The ability of an LNA modified oligo (5′-GGTGGTTTGTTTG-3′, LNAnucleosides in bold) to serve as primer in template dependent, enzymaticelongation were investigated with 3 different classes of polymerases. Areverse transcriptase M-MuLV (Boehringer Mannheim) which can use bothRNA and DNA as template, the Klenow polymerase which is representativeof standard DNA polymerases and a thermostable polymerase, BM-TAQ(Boehringer Mannheim). As control the extension reactions were conductedusing the identical unmodified DNA primer (5′-GGTGGTTTGTTTG-3′). The LNAand DNA primers were labelled with ³²P-γ-ATP as previously described inExample 137. A 50mer DNA oligo(5′-AAAAATCGACGCTCAAGTCAGAAAAGCATCTCACAAACAAACAAACCACC-3′) was used astemplate. The reaction with M-MuLV (Boehringer Mannheim,) contained 2 μlof either labelled LNA-primer or DNA primer (10 μM), 2 μl of DNAtemplate (10 μM), 2 μl of 2 mM dNTP, 2 μl of 10× buffer (500 mMTris-HCl, 300 mM KCl, 60 mM MgCl₂, 100 mM DTT, pH 8.3 (37° C.)), 1 μl ofenzyme (20 U/μl) and water to 20 μl. The reactions were incubated at 37°C. for 60 min. The reaction with Klenow polymerase (USB) contained 2 μlof either labelled LNA or DNA primer (10 μM), 2 μl of DNA template (10μM), 2 μl of 2 mM dNTP, 2 μl of 10× buffer (100 mM Tris-HCl, 50 mMMgCl₂, 75 mM DTT, pH 7.5), 1 μl of enzyme (10 U/μl) and water to 20 μl.The reactions were incubated at 37° C. for 60 min. The reaction withBM-Taq (Boehringer Mannheim) contained 2 μl of either labelled LNA orDNA-primer (10 μM), 2 μl of DNA template (10 μM), 2 μl of 2 mM dNTP, 2μl of 10× buffer (100 mM Tris-HCl, 15 mM MgCl₂, 50 mM KCL, pH 8.3), 1 μlof enzyme (5 U/μl) and water to 20 μl. The reactions were incubated at astarting temperature of 37° C. and ramped at 1° C./min to 60° C. wherethey were maintained for 30 min. At the end of the incubation period thereactions were stopped by the addition of 10 μl of loading buffer (0.25%(w/v) bromophenol blue, 0.25% (w/v) xylene cyanol, 80% (v/v) formamid).The samples were heated to 95° C. for 1 min., placed on ice and 2 μl wasloaded onto a 8% sequencing polyacrylamide gel and electrophoresed on aLife Technologies Inc. BRL model 52. After electrophoresis the gel wasdried on the glass plate and subjected to autoradiography (X-ray film:Kodak X-Omat AR). As shown in FIG. 7, clear and similar extensionproducts are observed with both the LNA and DNA primer when either theKlenow polymerase (lanes 3) or the BM-Taq polymerase (lanes 5) is used.When M-MuLV reverse transcriptase is used (lanes 2) an extension productcan be detected only in the case of the LNA-primer. The labelled LNA andDNA primer that have not been subjected to enzymatic elongation arepresent in lanes 1, 4 and 6. We conclude that the incorporation of LNAnucleosides into standard DNA oligos does not prevent recognition of theoligo/template duplex by nucleic acid polymerases. We further concludethat LNA modified oligos act as efficiently as primers as unmodified DNAoligos.

Example 148

LNA modified oligo functions as primers in target amplificationprocesses. The ability of LNA modified oligos to act as primers in PCRamplification was analysed with three oligos differing only in thenumber of LNA nucleosides they contained: 4 LNA nucleosides (AL2 primer:5′-GGTGGTTTGTTTG-3′, LNA nucleosides in bold), 1 LNA nucleoside (AL10primer: 5′-GGTGGTTTGTTTG-3′, LNA nucleoside in bold) and no LNAnucleoside (FP2 primer: 5′-GGTGGTTTGTTTG-3′). The PCR reactions (1000contained either no template (control), 0.01 ng, 0.1 ng or 1 ng oftemplate (pUC19 plasmid), 0.2 μM reverse primer(5′-GTGGTTCGCTCCAAGCTG-3′), 0.2 μM of either the AL2, AL10 or FP2forward primer, 200 μM of dATP, dGTP, dCTP and dTTP, 10 mM Tris-HCl pH8.3, 1.5 mM MgCl₂, 50 mM KCl and 2.5 U of the BM-Taq polymerase. A totalof 50 cycles each consisting of 94° C. 1 min.-45° C. 1 min.-72° C. 1.5min. were conducted (with an additional 2.5 U of Taq polymerase addedafter the first 30 cycles) on a Techne Genius thermocycler. After thefinal cycle the reactions were incubated at 72° C. 3 min. and then at 4°C. overnight. To 30 μl of each reaction was added 6 μl of loading buffer(0.25% (w/v) bromophenol blue and 40% (v/v) glycerol) and the samples(together with a Amplisize™ size marker) were loaded onto a 2% agarosegel and electrophoresed for 45 min. at 150V. Finally, the gel wasstained with ethidiumbromid and photographed. As shown in FIG. 8 the PCRreactions using the unmodified forward primer FP2 and unmodified reverseprimer generates detectable amplicons of the correct sizes with allamounts of template used (lane 9: 0.01 ng template, lane 10: 0.1 ng andlane 11: 1 ng). No signal is obtained in the control reaction withouttemplate (lane 12). When the FP2 forward primer is replaced by theprimer containing 1 central LNA nucleoside (AL10) amplicons are alsodetected with all amounts of template used (lane 5: 0.01 ng, lane 6: 0.1ng and lane 7: 1 ng). This clearly indicates that the AL10 primersustains an exponential amplification. i.e. the AL10 primer can be bothextended and used as template in its entirety. Again, the controlreaction without template (lane 8) does not produce an amplicon. Whenthe FP2 forward primer is replaced by the primer containing 4 centralLNA nucleosides (AL2), amplicons of the correct size cannot be detectedin any of the reactions. (lane 1: 0.01 ng template, lane 2: 0.1 ng, lane3: 1 ng and lane 4: no template). With the highest concentration oftemplate (1 ng), however, a high molecular weight band appears in thegel (lane 3). This, however, is an artefact of the RP1 primer asindicated by the control reaction wherein each of the primers AL2 (laneA), AL10 (lane B), FP2 (lane C) and RP1 (lane D) were tested for theirability to produce an amplicon with the highest amount of template (1ng). Since AL2 was shown to act as a primer in Example 147, the absenceof detectable amplicons strongly indicates that it lacks the ability toact as a template, i.e. the block of 4 consecutive LNA nucleosidesblocks the advance of the polymerase thereby turning the reaction into alinear amplification (the product of which would not be detectable bythe experimental set-up used). We conclude that LNA modified oligos canbe used as primers in PCR amplification. We further conclude that thedegree of amplification (graded from fully exponential to linearamplification) can be controlled by the design of the LNA modifiedoligo. We note that the possibility to block the advance of thepolymerase by incorporating LNA nucleosides into the primer facilitatesthe generation of amplicons carrying single stranded ends. Such ends arereadily accessible to hybridisation without denaturation of the ampliconand this feature could be useful in many applications.

Example 149

An LNA modified oligomer carrying a 5′ anthraquinone can be covalentlyimmobilised on a solid support by irradiation and the immobilisedoligomer is efficient in the capture of a complementary DNA oligo.Either 25 μmol/μl or 12.5 μmol/μl of an anthraquinone DNA oligo(5′-AQ-CAG CAG TCG ACA GAG-3′) or an anthraquinone LNA modified DNAoligo (5′-AQ-CAG CAG TCG ACA GAG-3′; LNA monomer is underlined) wasspotted (1 μl/spot) in 0.2 M LiCl on a polycarbonate slide (Nunc). Theoligos were irradiated for 15 min with soft UV light. After irradiationthe slide was washed three times in Milli-Q water and air-dried. 25 mlof 0.5 μmol/μl of complimentary biotinylated oligomer (5′-biotin-CTC TGTCGA CTG CTG-3′) was hybridised to the immobilised oligomers in 5×SSCT(75 mM Citrate, 0.75 M NaCl, pH 7.0, 0.1% Tween 20) at 50° C. for 2hours. After washing four times with 1×SSCT and one time phosphatebuffered saline (PBST, 0.15 M Na⁺, pH 7.2, 0.05% Tween 20), 25 ml PBSTcontaining 0.06 μg/ml streptavidin conjugated horse radish peroxidaseand 1 μg/ml streptavidin were added to the slide. The slide wasincubated for 30 min and washed 4 times with 25 ml PBST. The slide wasvisualised by using chemoluminescent substrate (SuperSignal; Pierce) asdescribed by the manufacturer and X-ray film (CL-XPosure film, Pierce34075). As shown in FIG. 9 both the AQ-DNA oligo and the AQ-LNA modifiedDNA oligo yields a clearly detectable signal. We conclude thatanthraquinone linked LNA modified DNA oligos can be efficiently attachedto a solid surface by irradiation and that oligos attached in this waysare able to hybridise to their complementary target DNA oligos.

Example 150

Hybridisation and detection on an array with different LNA modifiedCy3-labelled 8mers. Slide preparation: Glass slides were aminosilanisedusing a 10% solution of amino propyl triethoxy silane in acetonefollowed by washing in acetone. The following oligonucleotides werespotted out onto the slides:

Oligo  Oligo Pens used sequence 1 + 2 + 3 Sequence cf. probes Seq. 35′-GTA TGG 1 pmol/μl 1 internal mismatch AG-3′ Seq. 6 5′-GTA TGA1 pmol/μl match AG-3′

Ten repeat spots, approximately 1 nl each spot, were performed for eacholigonucleotide from each pen on each of 12 slides.

Probes (LNA monomers in bold):

Seq. No. aZ1 5′-Cy3-CTT CAT AC-3′ Seq. No. aZ2 5′-Cy3-CTT CAT AC-3′Seq. No. aZ3 5′-Cy3-CTT CAT AC-3′ Seq. No. 16 5′-Cy3-CTT CAT AC-3′Slides and conditions for hybridisation:Slides 1, 2 and 3 hybridised with aZ1 probe @ 300 fmol/μl, 30 fmol/μl, 3fmol/μlSlides 4, 5 and 6 hybridised with aZ2 probe @ 300 fmol/μl, 30 fmol/μl, 3fmol/μlSlides 7, 8 and 9 hybridised with aZ3 probe @ 300 fmol/μl, 30 fmol/μl, 3fmol/μlSlides 10, 11 and 12 hybridised with seq. 16 probe @ 300 fmol/μl, 30fmol/3 fmol/μl

A probe diluted in 30 μl hybridisation buffer (5×SSC, 7% sodium laurylsarcosine) was pipetted along the length of each slide, covered with acoverslip, and placed into a plastic box on top of a plastic insert,which was lying on a paper towel wetted with water. Boxes were coveredwith aluminium foil to keep light out, and incubated at +4° C.overnight.

Slide Washes: Coverslips were removed and the slides inserted in racks(6 slides per rack) which were placed in glass slide dishes, wrapped infoil:

Slide Probe Number Wash buffer (4° C.) Wash time sequence 1, 2, 3 5 ×SSC, 0.1% Tween-20 2 × 5 min Seq. No. aZ1 4, 5, 6 5 × SSC, 0.1% Tween-202 × 5 min Seq. No. aZ2 7, 8, 9 5 × SSC, 0.1% Tween-20 2 × 5 min Seq. No.aZ3 10, 11, 12 5 × SSC, 0.1% Tween-20 2 × 5 min Seq. No. 16After washing, slides were blow-dried and scanned. The fluorescence wasimaged on a slide scanner and the data analysed from ImageQuant software(Molecular Dynamics). As shown in FIG. 11, no binding of the Cy3labelled probes is observed to the mismatched oligo 3 with either theunmodified probe (slide 10-12), single LNA modified probe aZ1 (slide1-3) single LNA modified probe aZ2 (slide 4-6) or triple LNA modifiedprobe aZ3 (slide 7-9) (i.e. the obtained signal with the mismatchedoligo 3 is comparable to the background signal). With complementaryoligo 6, specific signals are observed in all cases. The intensity ofthese signals clearly correlates with the number of LNAs present in theprobes and with the concentration of the probes. Each LNA T residueapproximately increased the signal strength by about a factor of 2 overthat of the normal DNA oligo probe, i.e. aZ1 and aZ2=2× signal ofsequence 16, and aZ3=8× signal of sequence 16. The match/mismatchdiscrimination is good with the LNA T base replacements, and with theincreased signal strength, the mismatch discriminations appear to beeasier.

Example 151

Hybridisation and detection of end mismatches on an array with LNAmodified Cy3-labelled 8mers. Slide preparation: Glass slides wereaminosilanised using a 10% solution of amino propyl triethoxy silane inacetone followed by washing in acetone. The following oligonucleotideswere spotted out at 1 pmol/μl onto the slides:

Seq No. 9 5′-GTGTGGAG-3′ Seq No. 15 5′-GTGTGGAA-3′ Seq No. 1315′-GTGTGGAT-3′ Seq No. 132 5′-GTGTGGAC-3′ Seq No. 133 5′-ATGTGGAA-3′Seq No. 134 5′-CTGTGGAA-3′ Seq No. 135 5′-TTGTGGAA-3′

Ten repeat spots, approximately 1 nl each spot, were performed for eacholigonucleotide from each of 6 pens on each of 12 slides.

Probes (LNA monomers in bold):

DNA Probe No.1: 5′-Cy3-TTCCACAC-3′ Probe No.2: 5′-Cy3-GTCCACAC-3′Probe No.3: 5′-Cy3-ATCCACAC-3′ Probe No.4: 5′-Cy3-CTCCACAC-3′Probe No.5: 5′-Cy3-TTCCACAT-3′ Probe No.6: 5′-Cy3-TTCCACAG-3′ LNAProbe No.35Z-1: 5′-Cy3-TTCCACAC-3′ Probe No.35Z-2: 5′-Cy3-GTCCACAC-3′Probe No.35Z-3: 5′-Cy3-ATCCACAC-3′ Probe No.35Z-4: 5′-Cy3-CTCCACAC-3′Probe No.35Z-5: 5′-Cy3-TTCCACAT-3′ Probe No.35Z-6: 5′-Cy3-TTCCACAG-3′

Probes with LNA monomers are prefixed with 35Z- as part of the sequencenumber. Specific LNA monomers are indicated in italics/bold and aresituated at the 3′ and 5′ ends of the LNA oligos.

Slides and conditions for hybridisation: Each probe sequence washybridised on a separate slide, and all probe concentrations were 1fmol/μl. Each probe was diluted in hybridisation buffer (5×SSC, 7%sodium lauryl sarcosine), of which 30 μl was pipetted along the lengthof each slide, covered with a coverslip, and placed into a plastic boxon top of a plastic insert, which was lying on a paper towel wetted withwater. Boxes were covered with aluminium foil to keep light out, andincubated at +4° C. overnight.

Slide Washes: Coverslips were removed and the slides inserted in racks(8 slides per rack) which were placed in glass slide dishes, wrapped infoil. All slides were washed in 5×SSC for 2×5 min at +4° C. Afterwashing, slides were blow-dried and scanned. The fluorescence was imagedon a slide scanner and the data analyzed from ImageQuant software(Molecular Dynamics).

Conclusions: As shown in FIGS. 12 and 13, probes containing LNAnucleosides at their 5′ and 3′ ends are in the majority of casessignificantly better in discriminating between matched and mismatchedtarget sequences than their corresponding unmodified oligonucleotides.

For DNA oligos, C=T mismatches were the most difficult to distinguish,for example, where probe sequence 1 hybridised to target sequence 132and where probe sequence 5 hybridised to target sequence 134. Othermismatches were visible such as T=T and G=T mismatches, but these spotswere less intense, for example where probe sequences 5 and 6respectively hybridised to target sequence 135. The LNA oligos,significantly reduced these C=T and T=T mismatch spot intensites, tocomparable levels to other mismatches. The relative spot intensities ofprobe sequences 1, 2 and 3 were similar for the DNA and LNA oligos.However, with probe sequences 4, 5 and 6, the LNA oligos gave asignificantly increased spot intensity when hybridised to their matchtarget sequences 9, 133 and 134 respectively.

Example 152

Hybridization and detection of end mismatches on an array with AT andall LNA modified Cy3-labelled 8mers. Slide preparation: Glass slideswere aminosilanized using a 10% solution of amino propyl triethoxysilane in acetone followed by washing in acetone. The followingoligonucleotides were spotted out at 1 pmol/μl onto the slides:

Seq No. 9 5′-GTGTGGAG-3′ Seq No. 15 5′-GTGTGGAA-3′ Seq No. 1315′-GTGTGGAT-3′ Seq No. 132 5′-GTGTGGAC-3′ Seq No. 133 5′-ATGTGGAA-3′Seq No. 134 5′-CTGTGGAA-3′ Seq No. 135 5′-TTGTGGAA-3′

Ten repeat spots, approximately 1 nl each spot, were performed for eacholigonucleotide from each of 6 pens on each of 36 slides.

Probes: (LNA monomers in bold):

DNA: Probe No.1: 5′-Cy3-TTCCACAC-3′ Probe No.2: 5′-Cy3-GTCCACAC-3′Probe No.3: 5′-Cy3-ATCCACAC-3′ Probe No.4: 5′-Cy3-CTCCACAC-3′Probe No.5: 5′-Cy3-TTCCACAT-3′ Probe No.6: 5′-Cy3-TTCCACAG-3′ AT LNA:Probe No.ATZ-1: 5′-Cy3-TTCCACAC-3′ Probe No.ATZ-2: 5′-Cy3-GTCCACAC-3′Probe No.ATZ-3: 5′-Cy3-ATCCACAC-3′ Probe No.ATZ-4: 5′-Cy3-CTCCACAC-3′Probe No.ATZ-5: 5′-Cy3-TTCCACAT-3′ Probe No.ATZ-6: 5′-Cy3-TTCCACAG-3′A11 LNA: Probe No.A11Z-1: 5′-Cy3-TTCCACAC-3′ Probe No.A11Z-2:5′-Cy3-GTCCACAC-3′ Probe No.A11Z-3: 5′-Cy3-ATCCACAC-3′ Probe No.A11Z-4:5′-Cy3-CTCCACAC-3′ Probe No.A11Z-5: 5′-Cy3-TTCCACAT-3′ Probe No.A11Z-6:5′-Cy3-TTCCACAG-3′

Probes with LNA monomers are prefixed with ATZ- or AllZ- as part of thesequence number. Specific LNA monomers are indicated in italics for theLNA oligos.

Slides and conditions for hybridization: Each probe sequence washybridized on a separate slide, and all probe concentrations were 1fmol/μl. Each probe was diluted in hybridization buffer (5×SSC, 7%sodium lauryl sarcosine), of which 30 μl was pipetted along the lengthof each slide, covered with a coverslip, and placed into a plastic boxon top of a plastic insert, which was lying on a paper towel wetted withwater. Boxes were covered with aluminium foil to keep light out, andincubated at room temperature overnight.

Slide Washes: Coverslips were removed and the slides inserted in racks(9 slides per rack) which were placed in glass slide dishes, wrapped infoil.

All slides were washed in 5×SSC for 2×5 minutes at RT. After washing,slides were blow-dried and scanned. The fluorescence was imaged on aslide scanner and the data analyzed from ImageQuant software (MolecularDynamics).

Conclusion: As shown in FIGS. 15A, 15B and 15C, The average intensity ofDNA hybridization at room temperature was about 10% of the intensityachieved with the AT or all LNA modified oligos. No spots were seen onslides hybridized with DNA probes 5 and 6. These conditions weretherefore not optimal for the DNA probes. However, the match/mismatchdiscrimination is very good with the LNA nucleoside replacements at theA and T bases. The stringency for the all LNA oligos may not be greatenough as the match/mismatch discrimination was not as good as for theAT LNA oligos.

The oligos with LNA modifications worked very well, and the mismatchesthat were the most difficult to discriminate were;

Probe 1 to target 135=CT mismatch

Probe 2 to target 131=GT mismatch

Probe 3 to target 15=AA mismatch

Probe 4 to target 131=CT mismatch

Probe 5 to target 135=TT mismatch

Probe 6 to target 135=GT mismatch

Probe 6 to target 133=GA mismatch

The AT LNA oligos gave good discrimination where these mismatch spotintensities were typically at the most 50% of the intensity of the matchspots. For these mismatches, the all LNA oligos gave mismatch spotintensities about 50 to 70% of the match spot intensities. Overall, LNAmodifications allows the use of higher temperatures for hybridizationsand washes, and end mismatches can be discriminated. These results areat least as good as those from DNA probes hybridised at 4° C. (seeexample 151).

Example 153

Use of [α³³P] ddNTP's and ThermoSequenase™ DNA Polymerase to SequenceDNA Templates Containing LNA T Monomers. Radiolabelled terminatorsequencing reactions were set up in order to test the ability of the LNAT monomer to be accepted as a template for DNA polymerases. The 15merprimer (sequence: 5′-TGC ATG TGC TGG AGA-3′) was used to prime thefollowing short oligonucleotide sequences (LNA monomer in bold):

Template 1 3′- ACG TAC ACG ACC TCT ACC TTG CTA -5′ TemplateTZ13′- ACG TAC ACG ACC TCT ACC TTG CTA -5′

The following reaction mixes were made:

Template 1 mix:

2 μl x16 ThermoSequenase Buffer 6 μl Primer 2 pmole/μl 6 μl Template 1 1pmole/μl 4 μl Water 2 μl ThermoSequenase DNA Polymerase (4 U/μl) 20 μl Total volumeTemplate TZ1 mix

2 μl x16 ThermoSequenase Buffer 6 μl Primer 2 pmole/μl 6 μl Template TZ11 pmole/μl 4 μl Water 2 μl ThermoSequenase DNA Polymerase (4 U/μl) 20μl  Total volume

2 μl Nucleotide mix (7.5 μM each dNTP) was added to each of 8 Eppendorftubes. 0.5 μl [α³³P] ddATP was added to tubes 1 and 5. 0.5 μl [α³³P]ddCTP was added to tubes 2 and 6. 0.5 μl [α³³P] ddGTP was added to Tubes3 and 7. 0.5 μl [α³³P] ddTTP was added to tubes 4 and 8. 4.5 μl ofTemplate 1 mix was added to each of tubes 1-4. 4.5 μl of Template TZ1mix was added to each of tubes 5-8. All the reactions were incubated at60° C. for 3 min. The reactions were stopped by the addition of 4 μlformamide/EDTA stop solution. Reactions were heated at 95° C. for 3 minbefore loading onto a 19% polyacrylamide 7M urea gel. The gel was fixedin 10% acetic acid 10% methanol before transferring to 3MM paper anddrying. The dried gel was exposed to Kodak Biomax autoradiography film.

The results are depicted in FIG. 18 (track 1-4) and FIG. 19 (5-8). Thetracks correspond to the following reactions: (FIG. 18): Lane 1-ddATPtrack. Lane 2-ddCTP track. Lane 3-ddGTP track, Lane 4-ddTTP track. Lane5-8-32 base oligo markers; FIG. 19: Lane A-8-32 base oligo markers. Lane5-ddATP track. Lane 6-ddCTP track. Lane 7-ddGTP track. Lane 8-ddTTPtrack.

As is evident from FIGS. 18 and 19, the full sequence of both templatescan easily be read from the autorad. The sequence is 5′-TGG AAC GTA-3′which corresponds to the template sequence 3′-ACC TTG CTA-5′. This showsthat a single LNA T monomer can act as a template for DNA polymerases.The LNA T monomer is specifically copied as “T” with ddATP beingincorporated.

Therapeutic Applications

Example 154

LNA modified oligos can be transferred into cells. Experiment withradiolabelled LNA oligos. 10 pmol of a oligodeoxynucleotide (ODN)(ODN#10: 5′-TTA ACG TAG GTG CTG GAC TTG TCG CTG TTG TAC TT-3′, a 35-mercomplementary to human Cathepsin D) and 10 pmoles of two LNA oligos:AL16 (5′-d(TGT GTG AAA TTG TTA T)-3′, LNA nucleosides in bold) and AL17(5′-d(ATA AAG TGT AAA G)-3′, LNA nucleosides in bold) were mixed with T4polynucleotide Kinase (10 units, BRL cat. no. 510-8004SA), 5 μlgamma-³²P-ATP 5000 Ci/mmol, 10 uCi/μl (Amersham) in kinase buffer (50 mMTris/HCl pH 7.6, 10 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA). The samples wereincubated for 45 min at 37° C. and afterwards heated to 68° C. for 10min, and then moved to +0° C. Unincorporated nucleotides were removed bypassage over Chroma Spin TE-10 columns (Clontech cat. no. K1320-1). Theyields were 5×10⁵ cpm/μl, 2×10⁵ cpm/μl and 0.8×10⁵ cpm/μl for ODN#10,AL16 and AL17, respectively. MCF-7 human breast cancer cells originallyobtained from the Human Cell Culture Bank (Mason Research Institute,Rockville) were cultured in DME/F12 culture medium (1:1) supplementedwith 1% heat inactivated fetal calf serum (Gibco BRL), 6 ng/ml bovineinsulin (Novo) and 2.5 mM glutamax (Life Technologies) in 25 cm² cellculture flasks (Nunclon, NUNC) and incubated in a hunified incubator at37° C., 5% CO₂, 20% O₂, 75% N₂. The MCF-7 cells were approximately 40%confluent at the time of the experiment. A small amount (less than 0.1μmol) of the kinased oligos were mixed with 1.5 μg pEGFP-NI plasmid(Clontech cat. no. 60851) and mixed with 100 μl diluted FuGENE6transfection agent (Boehringer Mannheim cat no. 1 814 443), dilution: 5μl FuGENE6 in 95 μl DME/F12 culture medium without serum. TheFuGENE6/DNA/oligo-mixture were added directly to the culture medium (5ml) of adherent growing MCF-7 cells and incubated with the cells for 18hours, closely following the manufacturers directions. Three types ofexperiments were set up. 1) ODN#10+pEGFP-NI; 2) AL16+pEGFP-NI; 3)AL17+pEGFP-NI. Cellular uptake of DNA/LNA material were studied byremoving FuGENE6/DNA/oligo-mixture containing medium (an aliquot wastransferred to a scintillator vial). Cells were rinced once withphosphate buffered saline (PBS), fresh culture medium was added andcells inspected by fluorescence microscopy. Approximately 30% of thetransfected cells contained green fluorescent material, indicating thatapproximately 30% of the cells have taken up the pEGFP-NI plasmid andexpressed the green fluorescent protein coded by this plasmid. Followingfluorescence microscopy the adherent MCF-7 cells were removed from theculture flasks. Briefly, the culture medium was removed, then cells wererinsed with a solution of 0.25% trypsin (Gibco BRL) 1 mM EDTA in PBS(without Mg²⁺ and Ca²⁺), 1 ml trypsin/EDTA was added and cells wereincubated 10 min at 37° C. During the incubation the cells loosened andwere easily resuspended and transferred to scintillator vials. The cellswere then completely dissolved by addition of 10 ml Optifluorscintillation coctail (Packard cat. no. 6013199), and the vials werecounted in a Wallac 1409 scintillation counter. The results were asfollows: 1) ODN#10+pEGFP-NI: approximately 1.4% of the addedradioactivity were associated with cellular material; 2) AL16+pEGFP-NI:approximately 0.8% of the added radioactivity were associated withcellular material; and 3) AL17+pEGFP-NI: approximately 0.4% of the addedradioactivity were associated with cellular material. We conclude that0.4-0.8% of the added LNA oligos were taken up by the cells.

Example 155

LNA is efficiently delivered to living human MCF-7 breast cancer cells.To increase the efficiency of LNA-uptake by human MCF-7 cells differenttransfection agents were tested with various concentrations of5′FITC-labelled LNAs and DNA. The oligonucleotides described in thetable below were tested.

TABLE Oligonucleotides tested Sequence Name (LNA monomers in bold)Characteristics AL16 5′-TGT GTG AAA TTG TTA T-3′ LNA, enzym. FITC-labeled AL17 5′-ATA AAG TGT AAA G-3′ LNA, enzym.  FITC-labeledEQ3009-01 5′-TGC CTG CAG GTC GAC T-3′ LNA-FITC- labeled EQ3008-015′-TGC CTG CAG GTC GAC T-3′ DNA-FITC- labeled

AL16 and AL17 were enzymatically labelled with FITC as described inExample 128. EQ3009-01 and EQ3008-01 were labelled with FITC by standardsolid phase chemistry. Three transfection agents were tested: FuGENE-6(Boehringer Mannheim cat. no. 1 814 443), SuperFect (Quiagen cat. no.301305) and Lipofectin (GibcoBRL cat. no. 18292-011). Human MCF-7 breastcancer cells were cultured as described previously (Example 154). Threedays before the experiments the cells were seeded at a cell density ofapprox. 0.8×10⁴ cells per cm². Depending on the type of experiment theMCF-7 cells were seeded in standard T25 flasks (Nunc, LifeTechnologiescat. no. 163371A), 24 wells multidish (Nunc, LifeTechnologies cat. no.143982A) or slide flasks (Nunc, LifeTechnologies cat. no. 170920A). Theexperiments were performed when cells were 30-40% confluent. Cellularuptake of LNA and DNA was studied at serum-free conditions, i.e. thenormal serum containing DME/F12 medium was removed and replaced withDME/F12 without serum before the transfection-mixture was added to thecells. Under these conditions SuperFect proved to be toxic to the MCF-7cells. Transfection mixtures consisting of SuperFect and either plasmidDNA (pEGFP-N1, Clontech cat. no. 6085-1), oligo DNA or oligo LNA wasequally toxic to MCF-7 cells. In contrast to SuperFect, FuGene6 andLipofectin worked well with plasmid DNA (pEGFP-N1). However, onlylipofectin was capable of efficient delivery of oligonucleotides toliving MCF-7. Briefly, efficient delivery of FITC-labelled LNA and DNAto MCF-7 cells was obtained by culturing the cells in DME/F12 with 1%FCS to approx. 40% confluence. The Lipofectin reagent was then diluted40× in DME/F12 medium without serum and combined with the oligo to aconcentration of 750 nM oligo. The oligo-Lipofectin complex was allowedto form for 15 min at r.t., and further diluted with serum-free mediumto at final concentration of 250 nM oligo, 0.8 ug/ml Lipofectin. Then,the medium was removed from the cells and replaced with the mediumcontaining oligo-Lipofectin complex. The cells were incubated at 37° C.for 6 hours, rinsed once with DME/F12 medium without serum and incubatedfor a further 18 hours in DME/F12 with 1% FCS at 37° C. The result ofthe experiment was evaluated either directly on living cells in cultureflasks or in 24 wells multidishes or on cells cultured in slide flasksand fixed in 4% ice-cold PFA. In all cases a Leica DMRB fluorescencemicroscope equipped with a high resolution CCD camera was used. Theresult with living cells is shown in FIG. 16 and the result with fixedcells cultured in slide flask is shown in FIG. 17. Both the cells inFIGS. 16 and 17 was transfected with the FITC-labelled AL16 LNAmolecule. By counting total number of cells and green fluorescent cellsin several fields we observe that FITC-labelled AL16 LNA was transfectedinto approximately 35% of the MCF-7 cells. Importantly, we saw that theLNA predominantly was localised in the nuclei of the cells (FIG. 17).This is noteworthy, since nuclear uptake of fluorescent oligoscorrelates with their antisense activity (Stein C. A. et al. (1997)Making sense of antisense: A debate. In HMS Beagle: A BioMedNetPublication (http://hmsbeagle.com/06/cutedge/overwiev.htm)). Increasingthe amount of oligo and lipofectin up to a final concentration of 1250nM oligo and 4 ug/ml lipofectin only increased the percentage of greenfluorescent cells marginally. Increasing the concentration even furtherwas toxic for the cells. Similar results were obtained with the otherLNAs and the FITC-labelled oligo DNA (see the table above). We concludethat: 1) LNA can be efficiently delivered to living MCF-7 breast cancercells by Lipofectin-mediated transfection. 2) A consistent highfraction, 30% or more of cells, is transfected using a finalconcentration of 250 nM LNA, 0.8 ug Lipofectin pr. ml growth mediumwithout serum. Increasing the concentrations of LNA and Lipofectin up to5 times only increased the transfection yield marginally. 3) Theprocedure transfected the LNA into the nuclei of the cells, whichaccording to literature is a good indication that such transfected LNAsmay exhibit antisense effects on cells.

Example 156

LNA modified oligos can be transferred into cells. Experiment withfluorescein labelled LNA oligos. Two LNA oligos: AL16 (5′-TGT GTG AAATTG TTA T-3′, LNA nucleosides in bold) and AL17 (5′-ATA AAG TGT AAAG-3′, LNA nucleosides in bold) were labeled with fluorescein asdescribed in Example 128. MCF-7 human breast cancer cells were culturedas described in Example 154. Three types of experiments were set up. 1)approximately 1.5 μg FITC-labelled AL16; 2) approximately 1.5 μgFITC-labelled AL17; and 3) approximately 0.75 μg FITC-labelled AL16 and0.75 μg pRSVβgal plasmid (a plasmid expressing the bacterial lac Z genecoded enzyme β-galactosidase, Tulchinsky et. al. (1992) PNAS, 89,9146-50). The two LNA oligos and the LNA-plasmid mix were mixed withFuGENE6 and added to MCF-7 cells as described in Example 154. Afterincubation for 18 hours cellular uptake of the LNA oligos were assessedby fluorescence microscopy of the cell cultures. A part of the treatedcells contained green fluorescent material (see FIG. 16), indicatingthat cells take up the fluorescein labelled LNA. The fluoresceinlabelled AL16 appeared superior to fluorescein labelled AL17 in thisrespect. After fluorescence microscopy the culture medium were removedfrom the cells treated both with fluorescein labelled AL16 and pRSVβgal.The cells were washed once with PBS, fixed in 2% (v/v) formaldehyde,0.2% (v/v) glutaraldehyde at 4° C. for 5 min and β-galactosidasecontaining cells were stained blue with X-gal (5-bromo-4-chloro-3-indoylβ-D-galactopyranosid) which turns from colorless to blue in the presenceof β-galactosidase activity. The X-gal staining showed that the pRSVβgaleffectively had been transferred into cells. We conclude that thefluorescein LNA oligos were taken up by the cells.

Example 157

LNA modified oligos are relatively stable under cell culture conditions.Following fluorescence microscopy as described in Example 156 cellstreated only with the fluorescein labelled AL16 LNA were allowed toincubate for an additional 3 days. During this period of time the numberof green fluorescent cells appeared unaltered. We conclude thatfluorescein labelled LNA oligos has a good stability under theconditions prevailing in cell culture.

Example 158

Blockade by Antisense Locked Nucleic Acids (LNA) of[D-Ala2]Deltorphin-Induced Antinociception in the Warm Water Tail FlickTest in Conscious Rats. Male Sprague-Dawley rats (300 g) were implantedwith an intrathecal (i.th). polyethylene catheter and allowed to recoverfor at least 5 days before start of injections (including controls). Theantisense LNA compounds (12.5 and 2.5 μg per injection) wereadministered in a 5 μl volume twice-daily (08.00 and 17.00 h) for 3days. No signs of non-specific effects or toxicity could be detected, asshown by observations of locomotor behavior and measurements of bodyweight. The day after the last injection the rats were injected with[D-Ala2]deltorphin (60 μg, i.th) and tested in the warm water (52° C.)tail flick test for δ opioid receptor-mediated antinociception. Data arepresented in FIG. 14 as medians based on 6-8 animals per group (dataconverted to percent maximum possible response, % MPE). Statisticalanalyses were performed by means of Kruskal-Wallis 1-way ANOVA by ranks,followed by comparisons of treatments versus control. As shown in FIG.14, deltorphin produced a robust antinociceptive effect insaline-treated controls. This response was statistically significantlysuppressed in both antisense LNA groups (12.5 and 2.5 μg) as comparedwith saline-treated controls.

LNA Solid Supports

Example 159

General method for DMT-LNA nucleoside succinates. Base protected DMT-LNAnucleoside and succinic anhydride (1.5 equivalents) were taken inanhydrous ethylene dichloride (˜10 ml/g of nucleoside). To the mixture,triethylamine (2 equivalents) was added and the mixture was stirred atroom temperature. Reaction was followed by HPLC (conditions same as fortritylation). After complete reaction (>95%), reaction mixture wasconcentrated, coevaporated with ethylene dichloride and acetonitrile,and dried in vacuo to remove triethylamine. Residue was dissolved inethylene dichloride or ethyl acetate (˜100 ml/g of starting nucleoside),washed with cold 10% citric acid (3×80 ml/g) and cold water (3×80 ml/g).Organic layer was dried over anhydrous sodium sulfate, filtered andconcentrated with or without addition of 1-2 equivalents oftriethylamine. Residual solid was coevaporated with anhydrousacetonitrile (2-3×) and dried in vacuo to give pure product as whitesolid.

General method for LNA nucleoside supports. Base protectedDMT-LNA-nucleoside succinate (free acid or triethylammonium salt, 65micromol/g of support), amino derivatised support (Primer Support™ 30HL,160 micromol amino groups/g of support), DMAP (3 mg/g of support) and1-(3-[dimethylamino]propyl)-3-ethylcarbodimide hydrochloride (80 mg/g ofsupport) were taken in anhydrous pyridine (6 ml/g of support). To thismixture, triethylamine (16 microliter/g of support) was added and themixture was kept on a shaker at 150 rpm overnight. Support was filtered,washed with methanol (3×10 ml/g of support) and dichloromethane (3×10ml/g of support). After air drying, support was dried in vacuo for 0.5h. To this 6% DMAP in anhydrous acetonitrile (Cap A, ˜3 ml/g of support)and a mixture of 20% acetic anhydride/30% 2,4,6-collidine/50%acetonitrile (Cap B, ˜3 ml/g of support) were added. The micture waskept on shaker for 5 h. Support was filtered, washed with anhydrousdichloromethane (2×10 ml/g of support) and dried as above. It wasresuspended in a mixture of Cap A and Cap B (total vol. 6 ml/g ofsupport) and kept on shaker overnight. Support was filtered, washed withmethanol (6×10 ml/g of support), dichloromethane (3×10 ml/g of support)and dried in air. It was further dried in vacuo for 5-6 h. Loading wasdetermined by dimethoxytrityl assay and was found to be approx. 40mmol/g.

Example 160

First Strand cDNA Synthesis Using Poly dT Primers Containing LNA Tmonomers. Reactions were set up in order to test the ability of poly dTprimers containing LNA T residues to prime 1^(st) strand cDNA synthesis.The following poly dT primers were tested (LNA monomers are in bold):

RTZ1 5′-TTT TTT TTT TTT TT-3′ RTZ2 5′-TTT TTT TTT TTT TT-3′ RTZ35′-TTT TTT TTT TTT TT-3′ RTZ4 5′-TTT TTT TTT TTT TT-3′ RTZ55′-TTT TTT TTT T-3′

Anchored poly dT primer from RPK0140 kit Cy Dye cDNA labelling kit(Amersham Pharmacia Biotech) was as a control.

Reactions were set up as follows for each of the primers above:

1 μl Arabidopsis mRNA 0.5 μg/μl 2 μl poly dT primer 8 pmoles/μl 4 μl x5AMV Reverse Transcriptase buffer 1 μl Water 8 μl Total volume

This mix was then heated to 75° C. for 3 min and then allowed to cool atroom temperature for at least 10 min.

The following was then added to each of the reactions:

1 μl 80 mM Sodium Pyrophosphate 1 μl Human Placental RibonucleaseInhibitor 20 U/μl 7 μl 0.5 mM dNTP solution 2 μl [α³³P] dATP 10 mCi/ml3000 Ci/mmole 1 μl AMV Reverse Transcriptase 20 U/μl 20 μl  Total volume

The reactions were incubated at 42° C. for 2 hours. The reactions werethen heated at 95° C. for 3 min before loading onto a 6% polyacrylamide7M urea gel. The gel was fixed in 10% acetic acid/10% methanol beforetransferring to 3mM paper and drying. The dried gel was exposed to KodakBiomax autoradiography film overnight.

The autoradiograph clearly showed that the LNA containingoligonucleotide primers RTZ1-4 were able to efficiently prime cDNAsynthesis. The amount and intensity of the cDNA products produced inthese reactions was equal to that produced with the anchored poly dTcontrol oligonucleotide. RTZ 5 did produce some cDNA, but the yield wassignificantly lower than that produced with the control oligo primer.

Example 161

LNA-modified oligonucleotides covalently attached to Separose beadsfunction efficiently in the sequence specific capture of RNA molecules.Three oligos were synthesised by chemistry (Amy Mueller) for evaluationin poly (rA) binding.

NH₂(T8)-T Control NH₂(T15)-T Control NH₂(LNA-T8)-T Test

200 nmol of each oligo were coupled to 50 mg of prepared CNBr-activatedSeparose 4B (Pharmacia) per booklet instructions. Unreacted bindingsites on the resin were blocked in 100 nM Tris pH 8.0.

Table of Oligo Binding Data T9 oligo T16 oligos LNA T9 oligo No oligoSteps A₂₆₀ units A₂₆₀ units A₂₆₀ units Control Oligo reacted 14.7 26.014.7 0 (200 nM) (200 nM) (200 nM) Unbound oligo 5.50 10.43 4.20 — ∴Boundoligo 9.20 15.57 10.50 — % Bound 62.6% 59.9% 71.4% —

Oligo bound resins were divided into two portions (˜25 mg resin each)for poly (rA) binding analysis in duplicate. Poly (rA) Pharmacia#27-4110-01 (dissolved at 28.2 A₂₆₀ units/ml in binding buffer) was usedfor binding. Five (5) A₂₆₀ units were bound to duplicate 25 mg portionsof each oligo bound resin per SOP QC 5543. Unbound “breakthrough” poly(rA) was quantitated by A₂₆₀ absorbance and used to calculate bound. Thefate of the bound poly (rA) was tracked through Low salt buffer wash andseveral elutions. As shown in Table 10 both the LNA and DNA coated beadsfunction efficiently in the capture of poly (rA) target molecules. TheLNA coated beads, however, bind the poly (rA) target much more tightlythan the DNA coated beads as evidenced by the poly (rA) elution profilesof the different beads. We conclude that 1) an LNA T9 oligo is efficientin the capture of RNA molecules containing a strech of A residues andthat 2) the captured RNA molecules are bound much more tightly to theLNA T9 oligo beads than to the control DNA T9 and DNA T16 oligo.

TABLE 1 Monomer Z T_(m)(° C.) T_(m) T_(m)(° C.) T_(m)(° C.) Na₂HPO₄/Oligo Target No. Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA TMAC 5′-d(GTGATATGC)-3′5′-d(GCATATCAC)-3′  1 28 42 5′-d(GCATTTCAC)-3′  2 12 315′-d(GCATGTCAC)-3′  3 19 23 5′-d(GCATCTCAC)-3′  4 11 305′-d(GCATAACAC)-3′  5 12 5′-d(GCATAGCAC)-3′  6 <10 5′-d(GCATACCAC)-3′  7<10 5′-(GCAUAUCAC)-3′  8 28 5′-(GCAUCUCAC)-3′  9 10 5′ -d(GTGATATGC5′d(GCATATCAC) 10 44 56 5′-d(GCATTTCAC) 11 27 43 5′-d(GCATGTCAC) 12 3043 5′-d(GCATCTCAC) 13 23 38 5′-d(GCATAACAC) 14 28 5′d(GCATAGCAC) 15 285′-d(GCATACCAC  15A 29 5′-(GCAUAUCAC) 16 50 5′-(GCAUCUCAC) 17 335′-d(GTGAGATGC)-3′ 5′-d(GCATATCAC)-3′ 18 26 39 5′-d(GCATTTCAC)-3′ 19 3344 5′-d(GCATGTCAC)-3′ 20 28 38 5′-d(GCATCTCAC)-3′ 21 49 575′-d(GCATAACAC)-3′ 22 <15 5′-d(GCATAGCAC)-3′ 23 <15 5′-d(GCATACCAC)-3′24 <15 5′-(GCAUAUCAC)-3′  24A 34 5′-(GCAUCUCAC)-3′  24B 595′-d(GTGAUATGC)-3′ 5′-d(GCATATCAC)-3′ 25 44 56 5′-d(GCATTTCAC)-3′ 26 2544 5′-d(GCATGTCAC)-3′ 27 32 43 5′-d(GCATCTCAC)-3′ 28 24 375′-d(GCATAACAC)-3′ 29 27 5′-d(GCATAGCAC)-3′ 30 28 5′-d(GCATACCAC)-3′ 3120 5′-d(GTGAGATGC)-3′ 5′-d(GCATATCAC)-3′ 32 17 34 5′-d(GCATTTCAC)-3′ 3316 30 5′-d(GCATGTCAC)-3′ 34 15 28 5′-d(GCATCTCAC)-3′ 35 33 445′-d(GCATAACAC)-3′ 36 9.0 5′-d(GCATAGCAC)-3′ 37 <5 5′-d(GCATACCAC)-3′ 38<5 5′-(GCAUCUCAC)-3′  38A 33 5′-d(GGTGGTTTGTTTG)-3′5′-d(CAAACAAACCACA)-3′ 39 31 47 55 5′-(CAAACAAACCACA)-3′  39A 32 525′-d(GGTGGTTTGTTTG)-3′ 5′-d(CAAACAAACCACA)-3′ 40 40 57 675′-(CAAACAAACCACA)-3′  40A 50 70 d(GGTGGTTTGTTTG)-3′5′-d(CAAACAAACCACA)-3′ 41 67 83 >90 5′-(CAAACAAACCACA)-3′  41A 85 >935′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 42 365′-(AAAAAAAAAAAAAA)-3′ 43 32 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 44 36 5′-(AAAAAAAAAAAAAA)-3′ 45 325′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 46 345′-(AAAAAAAAAAAAAA)-3′ 47 40 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 48 42 5′-(AAAAAAAAAAAAAA)-3′ 49 525′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 50 475′-(AAAAAAAAAAAAAA)-3′ 51 53 5′-d(TTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′52 80 5′-(AAAAAAAAAAAAAA)-3′ 53 70 5′-d(AAAACAAAA)-3′ 54 635′-d(AAAAGAAAA)-3′ 55 55 5′-d(AAAATAAAA)-3′ 56 65 5′-d(GTGAAATGC)-3′5′-d(GCATATCAC)-3′ 57 26 5′-d(GCATTTCAC)-3′ 58 45 5′-d(GCATGTCAC)-3′ 5923 5′-d(GCATCTCAC)-3′ 60 25 5′-d(GTGA^(Me)CATGC)-3′ 5′-d(GCATATCAC)-3′61 <15 5′-d(GTGA^(Me)CATGC)-3′ 63 32 5′-d(GCATATCAC)-3′5′-d(GCATTTCAC)-3′ 64 27 5′-d(GCATGTCAC)-3′ 65 53 5′-d(GCATCTCAC)-3′ 6632 5′-d(GTGACATGC)-3′ 5′-d(GCATATCAC)-3′ 67 32 5′-d(GCATGTCAC)-3′ 69 525′-d(GTGATATG Me C)-3′ 5′-d(GCATATCAC)-3′ 71 64 5′-d(GCATGTCAC)-3′ 73 525′-(GCAUAUCAC)-3′ 75 74 5′-(GCAUCUCAC)-3′ 76 60 5′-d(CACTATACG)-3′ 77 405′-d(GTGTTTTGC)-3′ 5′-d(GCAAAACAC)-3′ 78 52

TABLE 2 Monomer V T_(m) T_(m)(° C.) T_(m)(° C.) T_(m)(° C.) Oligo TargetNo. Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA Na₂HPO₄/TMAC 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 32 5′-(AAAAAAAAAAAAAA)-3′ 275′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 315′-(AAAAAAAAAAAAAA)-3′ 28 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 30 5′-(AAAAAAAAAAAAAA)-3′ 235′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 235′-(AAAAAAAAAAAAAA)-3′ 31 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 23 5′-(AAAAAAAAAAAAAA)-3′ 165′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ <105′-(AAAAAAAAAAAAAA)-3′ 42 5′-(AAAAAAGAAAAAAA)-3′ 37 5′-d(GTGATATGC)-3′5′-d(GCATATCAC)-3′ 26 5′-(GCAUAUCAC)-3′ 27

TABLE 3 Monomer X T_(m) T_(m)(° C.) T_(m)(° C.) T_(m)(° C.) Oligo TargetNo. Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA Na₂HPO₄/TMAC 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 23 5′-(AAAAAAAAAAAAAA)-3′ 235′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 195′-(AAAAAAAAAAAAAA)-3′ 23 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 9 5′-(AAAAAAAAAAAAAA)-3′ 155′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 5 5′-(AAAAAAAAAAAAAA)-3′14

TABLE 4 Monomer Y T_(m) T_(m)(° C.) T_(m)(° C.) T_(m)(° C.) Oligo TargetNo. Na₂HPO₄/EDTA Na₂HPO₄/NaCl/EDTA Na₂HPO₄/TMAC 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 36 5′-(AAAAAAAAAAAAAA)-3′ 375′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 355′-(AAAAAAAAAAAAAA)-3′ 37 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 35 5′-(AAAAAAAAAAAAAA)-3′ 365′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 325′-(AAAAAAAAAAAAAA)-3′ 33 5′-d(TTTTTTTTTTTTTT)-3′5′-d(AAAAAAAAAAAAAA)-3′ 36 5′-(AAAAAAAAAAAAAA)-3′ 365′-d(TTTTTTTTTTTTTT)-3′ 5′-d(AAAAAAAAAAAAAA)-3′ 585′-(AAAAAAAAAAAAAA)-3′ 58 5′-d(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 355′-(GCAUAUCAC)-3′ 35

TABLE 5 Monomer Z T_(m) Melting temperature (T_(m)/° C.) Oligo TargetNo. Y = A Y = C Y = T Y = G 5′-r(GTGATATGC)-3′ 5′-d(GCATYTCAC)-3′ 1 5534 38 37 5′-r(GUGAUAUGC)-3′ 5′-d(GCATYTCAC)-3′ 2 27  <10 <10 <105′-r(GTGATATGC)-3′ 5′-r(GCAUYUCAC)-3′ 3 63 45 — — 5′-r(GUGAUAUGC)-3′5′-r(GCAUYUCAC)-3′ 4 38 22 — —

TABLE 6 Monomer Z T_(m) Melting temperature Oligo Target No. (T_(m)/°C.) 5′-d(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 1 28 5′-d(GTGATATGC)-3′5′-d(GCATATCAC)-3′ 2 44 5′-d(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 3 405′-d(GTGATATGC)-3′ 5′-d(GCATATCAC)-3′ 4 63 5′-r(GTGATATGC)-3′5′-d(GCATATCAC)-3′ 5 74 5′-(GTGATATG Me C)-3′ 5′-d(GCATATCAC)-3′ 6 85

TABLE 7 Monomer Z (all-phosphoromonothioate oligonucleotides) T_(m)Melting temperature Oligo Target No. (T_(m)/° C.)5′-d(G^(S)T^(S)G^(S)A^(S)T^(S)A^(S)T^(S)G^(S)C)-3′ 5′-d(GCATATCAC)-3′ 121 5′-d(G^(S)T^(S)G^(S)A^(S)T^(S)A^(S)T^(S)G^(S)C)-3′ 5′-r(GCAUAUCAC)-3′2 17 5′-d(G^(S) T SG^(S)A^(S) T SA^(S) T SG^(S)C)-3′ 5′-d(GCATATCAC)-3′3 41 5′-d(G^(S) T SG^(S)A^(S) T SA^(S) T SG^(S)C)-3′ 5′-r(GCAUAUCAC)-3′4 47

TABLE 8 Monomer thio-Z (U^(S)) T_(m) Melting temperature Oligo TargetNo. (T_(m)/° C.) 5′-d(GTGAU sATGC)-3′ 5′-d(GCATATCAC)-3′ 1 34 5′-d(GTGAUsATGC)-3′ 5′-(GCAUAUCAC)-3′ 2 36 5′-d(GU sGAU sAU sGC)-3′5′-d(GCATATCAC)-3′ 3 42 5′-d(GU sGAU sAU sGC)-3′ 5′-(GCAUAUCAC)-3′ 4 525′-d(GTGTTTTGC)-3′ 5′-(GCAAAACAC)-3′ 5 27 5′-d(GU sGU s U s U s UsGC)-3′ 5′-d(GCAAAACAC)-3′ 6 51

TABLE 9 Monomers amino-Z (T^(NH)) and methylamino-Z (T^(NMe)) T_(m)Melting temperature Oligo Target No. (T_(m)/° C.) 5′-d(GTGAT NHATGC)-3′5′-d(GCATATCAC)-3′  1 33 5′-d(GTGAT NHATGC)-3′ 5′-(GCAUAUCAC)-3′  2 345′-d(GT NHGAT NHAT NHGC)-3′ 5′-d(GCATATCAC)-3′  3 39 5′-d(GT NHGAT NHATNHGC)-3′ 5′-(GCAUAUCAC)-3′  4 47 5′-d(GTGAT NMeATGC)-3′5′-d(GCATATCAC)-3′  5 33 5′-d(GTGAT NMeATGC)-3′ 5′-(GCAUAUCAC)-3′  6 365′-d(GT NMeGAT NMeAT NMeGC)-3′ 5′-d(GCATATCAC)-3′  7 39 5′-d(GT NMeGATNMeAT NMeGC)-3′ 5′-(GCAUAUCAC)-3′  8 49 5′-d(GT NMeGT NH T NMe T NH TNMeGC)-3′ 5′-d(GCAAAACAC)-3′  9 47 5′-d(GT NMeGT NH T NMe T NH TNMeGC)-3′ 5′-(GCAAAACAC)-3′ 10 63

TABLE 10 T9 oligo T16 oligos LNA T9 oligo No oligo Steps A₂₆₀ units A₂₆₀units A₂₆₀ units Control poly (rA) added 5.0/5.0 5.0/5.0 5.0/5.0 5.0/5.0poly (rA) breakthrough 1.75/1.61 1.84/1.78 1.83/1.82 5.09/5.14 ∴poly(rA) bound 3.25/3.39 3.16/3.22 3.17/3.18 0.0/0.0 % poly (rA) bound65.0%/67.8% 63.2%/64.4% 63.4%/63.6% 0.0%/0.0% Low Salt Wash/Elute0.24/0.24 0.11/0.12 .053/.055 0.14/0.13 TE Elute 15 min RT 2.37/2.720.83/0.93 0.02/0.04 0.01/0.02 TE Elute O.N. RT 0.38/0.37 1.76/1.690.11/0.07 .003/.004 TE Elute 30 min 65° C. .047/.040 0.38/0.46 1.62/1.70.005/.004 10 mM Tris pH 10 Elute .002/.002 0.03/0.03 0.10/0.10 0.01/0.011 mM HCl pH 4.0 Elute 0.07/0.06 0.06/0.04 0.26/0.23 0.01/0.01 Ave. A₂₆₀Recovered 3.20 3.14 2.18 — Ave. % A₂₆₀ Recovered 96.4% 98.4% 68.7% —

The invention claimed is:
 1. A method for using an oligomer to isolate,purify, amplify, detect, identify, quantify or capture a natural orsynthetic nucleic acid, the oligomer being modified with at least oneLocked Nucleic Acid (LNA), the method comprising the step of contactingLNA-modified oligomer with the nucleic acid under conditions sufficientto form a binding complex to isolate, purify, amplify, detect, identify,quantify or capture the nucleic acid, wherein the LNA is represented bythe following general formula I:

wherein X is selected from —O—; B is selected from hydrogen, hydroxy,optionally substituted C₁₋₄-alkoxy, optionally substituted C₁₋₄-alkyl,optionally substituted C₁₋₄-acyloxy, nucleobases, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands; P designates a radical positionfor an internucleoside linkage to a succeeding monomer or a 5′-terminalgroup, optionally including the substituent R⁵, one of the substituentsR², R²*, R³, and R³* is a group P* which designates an internucleosidelinkage or a 3′-terminal group; the substituents R⁴* and R²*, togetherdesignating the biradical —(CR*R*)_(r)—O—(CR*R*)_(s)—, each R* isindependently selected from hydrogen, halogen, hydroxy, mercapto, amino,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,DNA intercalators, photochemically active groups, thermochemicallyactive groups, chelating groups, reporter groups, and ligands, and r is0 (zero) and s is 1, or r is 1 and s is 0 (zero); and each of thesubstituents R¹*, R², R³, R⁵, and R⁵*, which are present and notinvolved in P or P* is independently selected from hydrogen, optionallysubstituted C₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl,optionally substituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy, carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and basic salts and acid addition salts thereof,wherein a natural or synthetic nucleic acid is isolated, purified,amplified, detected, identified, quantified, or captured using theoligomer.
 2. The method of claim 1, wherein the oligomer comprises 1 to1000 nucleosides of the formula I and 0-1000 nucleosides selected fromnaturally occurring nucleosides and nucleotide analogues, with theproviso that the sum of the number of nucleosides and the number ofLNA(s) is at least
 2. 3. The method of claim 1, wherein the oligomercomprises a photochemically active group, a thermochemically activegroup, a chelating group, a reporter group, or a ligand.
 4. The methodof claim 3, wherein the method further comprises the step ofimmobilizing the oligomer to a solid support.
 5. The method of claim 3,wherein the photochemically active group, thermochemically active group,chelating group, reporter group, or the ligand includes a spacer (K),said spacer comprising a chemically cleavable group.
 6. The method ofclaim 3, wherein the photochemically active group, the thermochemicallyactive group, the chelating group, the reporter group, or the ligand isattached via the biradical of at least one of the LNA(s) of theoligomer.
 7. The method of claim 1, wherein the method further comprisesthe steps of capturing and detecting the binding complex as beingindicative of presence of a double stranded or single stranded nucleicacid.
 8. The method of claim 7, wherein the nucleic acid is RNA or DNA.9. The method of claim 1, wherein the method further comprises the stepof purifying the nucleic acid from the binding complex.
 10. The methodof claim 9, wherein the nucleic acid is RNA or DNA.
 11. The method ofclaim 1, wherein formation of the binding complex is conducted by one ofin-situ hybridization, Southern hybridization, Dot blot hybridization,reverse Dot blot hybridization, or by Northern hybridization.
 12. Themethod of claim 1, wherein the binding complex serves as a template fora nucleic acid sequencing reaction, primer extension reaction, ornucleic acid amplification reaction.
 13. The method of claim 12, whereinthe nucleic acid amplification reaction is an essentially linearreaction.
 14. The method of claim 12, wherein the nucleic acidamplification reaction is an essentially exponential reaction.
 15. Themethod of claim 12, wherein the binding complex serves as a template fora nucleic acid amplification reaction, the method further comprising thestep of producing a double stranded DNA product comprising at least onesingle stranded end.
 16. The method of claim 1, wherein at least one ofthe LNA groups comprises a nucleobase as the substituent B.
 17. Themethod of claim 1, wherein one of the substituents R³ and R³* designatesP*.
 18. The method of claim 1, wherein one or more nucleosides have thefollowing formula Ia:

wherein P, P*, B, X, R¹*, R², R²*, R³, R³*, R⁴*, R⁵, and R⁵* are asdefined in claim
 1. 19. The method of claim 18, wherein R³* designatesP*.
 20. The method of claim 19, wherein the oligomer comprises onebiradical constituted by two non-geminal substituents.
 21. The method ofclaim 1, wherein R³* designates P*.
 22. The method of claim 21, whereinR² is selected from hydrogen, hydroxy, and optionally substitutedC₁₋₄-alkoxy, and R³*, R³, R⁵, and R⁵* designate hydrogen.
 23. The methodof claim 22, wherein B is a nucleobase.
 24. The method of claim 23,wherein B is selected from adenine, guanine, thymine, cytosine, uracil,purine, xanthine, diaminopurine, 8-oxo-N⁶-methyladenine,7-deazaxanthine, 7-deazaguanine, N⁴,N⁶-ethanocytosine,N⁴,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C₃-C₆)-alkynylcytosine, 2,6-diaminopyrimidino,2,6-diaminopyrazine,1-methyl-pyrazol[4,3-d]pyrimidine-5,7(4H,6H)-dione,1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione, 5-fluorouracil,5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine,isocytosine, isoguanine, and inosine.
 25. The method of claim 1, whereinany internucleoside linkage within the oligomer is selected fromlinkages consisting of 2 to 4 groups/atoms selected from —CH₂—, —O—,—S—, —NR^(H)—, >C═O, >NR^(H), >C═S, —Si(R″)₂— —SO—, —S(O)₂—, —P(O)₂—,—P(O,S)—, —P(S)₂—, —PO(R″)—, —PO(OCH₂)—, and —PO(NHR^(H))—, where R^(H)is selected from hydrogen and C₁₋₄-alkyl, and R″ is selected fromC₁₋₆-alkyl and phenyl.
 26. The method of claim 25, wherein theinternucleoside linkage is selected from —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—,—CH₂—CHOH—CH₂—, —O—CH₂—O, —O—CH₂—CH₂—, —O—CH₂—CH═, —CH₂—CH₂—O—,—NR^(H)—CH₂—, —CH₂—CH₂—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —NR^(H)—CO—O—,—NR^(H)—CO—NRH—, —NR^(H)—CS—NR^(H)—, —NR^(H)—C(═NR^(H))—NR^(H)—,—NR^(H)—CO—CH₂—NR^(H)—, —O—CO—O—, —O—CO—CH₂—O—, —O—CH₂—CO—O—,—CH₂—CO—NR^(H)—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—,—O—CH₂—CH₂—NR^(H)—, —CH═N—O—, —CH₂—NR^(H)—O—, —CH₂—O—N═, —CH₂—O—NR^(H)—,—CO—NR^(H)—CH₂—, —CH₂—NR^(H)—O—, —CH₂—NR^(H)—CO—, —O—NR^(H)—CH₂—,—O—NR^(H)—, —O—CH₂—S—, —S—CH₂—O—, —CH₂—CH₂—S—, —O—CH₂—CH₂—S—,—S—CH₂—CH═, —S—CH₂—CH₂—, —CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—S—CH₂—,—CH₂—SO—CH₂—, —CH₂—SO₂—CH₂—, —O—SO—O, —S(O)₂—O—, —O—S(O)₂—CH₂—,—O—S(O)₂—NR^(H)—, —NRH—S(O)₂—CH₂—, —O—S(O)₂—CH₂—, —O—P(O)₂—O—,—O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)—O—, —S—P(O,S)—O—, —S—P(S)₂—O—,—O—P(S)₂—S—, —S—P(O)₂—S—, —S—P(O,S)—S—, —S—P(S)₂—S, —O—PO(R″)—O—,—O—PO(OCH₃)—O—, —O—PO(BH₃)—O—, —O—PO(NHR^(N))—O—, O—P(O)₂—NR^(H)—,—NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, and —O—Si(R′)₂—O—.
 27. The methodof claim 26, wherein the internucleoside linkage is selected from—CH₂—O—NR^(H)—, —CH₂—NR^(H)—O—, —O—P(O)₂—, —O—P(O,S)—O—, —O—P(S)₂—O—,—NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, —O—PO(R″)—O—, and—O—PO(NHR^(N))—O—, where R^(H) is selected from hydrogen and C₁₋₄-alkyl,and R″ is selected from C₁₋₆-alkyl and phenyl.
 28. The method of claim1, wherein each of the substituents R¹*, R², R³, R³*, R⁵, and R⁵* of theone or more LNA nucleosides, which are present and not involved in P, P*is independently selected from hydrogen, optionally substitutedC₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, hydroxy, C₁₋₆-alkoxy,C₂₋₆-alkenyloxy, carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl,formyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, C₁₋₆-alkylcarbonylamino, carbamido,azido, C₁₋₆-alkanoyloxy, sulphono, sulphanyl, C₁₋₆-alkylthio, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, ligands, and halogen, wheretwo geminal substituents together may designate oxo, and where R^(N)*,when present and not involved in a biradical, is selected from hydrogenand C₁₋₄-alkyl.
 29. The method of claim 1, wherein each of thesubstituents R¹*, R², R³, R³*, R⁵, and R⁵* of the one or more LNAnucleosides which are present and not involved in P or P* designateshydrogen.
 30. The method of claim 1, wherein P is a 5′-terminal groupselected from hydrogen, hydroxy, optionally substituted C₁₋₆-alkyl,optionally substituted C₁₋₆-alkoxy, optionally substitutedC₁₋₆-alkylcarbonyloxy, optionally substituted aryloxy, monophosphate,diphosphate, triphosphate, and —W-A′, wherein W is selected from —O—,—S—, and —N(R^(H))— where R^(H) is selected from hydrogen andC₁₋₆-alkyl, and wherein, A′ is selected from DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands.
 31. The method of claim 1, whereinP* is a 3′-terminal group selected from hydrogen, hydroxy, optionallysubstituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkylcarbonyloxy,optionally substituted aryloxy, and —W-A′, wherein W is selected from—O—, —S—, and —N(R^(H))— where R^(H) is selected from hydrogen andC₁₋₆-alkyl, and where A′ is selected from DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands.
 32. The method of claim 1, whereinthe oligomer corresponds to the following formula V:G-[Nu-L]_(n(0))-{[LNA-L]_(m(q))-[Nu-L]_(n(q))}_(q)-G*  V wherein q is1-50; each of n(0) . . . , n(q) is independently 0-10000; each of m(1) .. . , m(q) is independently 1-10000; with the proviso that the sum ofn(0) . . . , n(q) and m(1) . . . , m(q) is 2-15000; G designates a5′-terminal group; each Nu independently designates a nucleosideselected from naturally occurring nucleosides and nucleoside analogues;each L independently designates an internucleoside linkage between twogroups selected from Nu and LNA, or L together with G* designates a3′-terminal group; and each LNA independently designates a nucleosideanalogue of the general formula I

wherein the substituents B, P, P*, R¹*, R², R²*, R³, R⁴*, R⁵, and R⁵*and X are as defined in claim
 1. 33. The method of claim 1, wherein theoligomer further comprises a PNA mono- or oligomer segment of theformula

wherein B is a defined above for the formula I, AASC designates hydrogenor an amino acid side chain, t is 1-5, and w is 1-50.
 34. The method ofclaim 1, wherein the oligomer has an increased specificity towardscomplementary ssRNA or ssDNA compared to a corresponding referenceoligomer which does not contain any LNA units.
 35. The method of claim1, wherein the oligomer has an increased affinity towards complementaryssRNA or ssDNA compared to a corresponding reference oligomer which doesnot contain any LNA units.
 36. The method of claim 1, wherein theoligomer is capable of binding to a target sequence in a dsDNA or dsRNAmolecule by of strand displacement or by triple helix formation.
 37. Themethod of claim 1, wherein the oligomer is more resistant to nucleasesthan a corresponding reference oligomer which does not contain any LNAunits.
 38. The method of claim 1, wherein the oligomer has nucleic acidcatalytic activity.
 39. The method of claim 1, wherein the oligomercomprises one or more 2′-O,4′-C-linked nucleoside units.
 40. A kit forthe isolation, purification, amplification, detection, identification,quantification, or capture of a natural or synthetic nucleic acid, thekit comprising a reaction body, at least one of the oligomers producedby claim 1; and directions for using the kit.
 41. The method of claim 1,wherein the oligomer comprises one or more nucleosides of the generalformula I wherein: the substituents R⁴* and R²*, together designatingthe biradical —(CR*R*)_(r)—O—(CRR*)_(s)—, each R* is independentlyselected from hydrogen, halogen, hydroxy, mercapto, amino, optionallysubstituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl, DNAintercalators, photochemically active groups, thermochemically activegroups, chelating groups, reporter groups, and ligands, and r is 0(zero) ands is greater than 1, or r is greater than 1 and s is 0 (zero).42. The method of claim 1, wherein the oligomer comprises one or morenucleosides of the general formula I wherein: B designates a nucleobase;P, optionally together with R⁵, designates a bond to the terminal end ofan internucleoside linkage to a succeeding monomer, one of thesubstituents R³ and R³* is a group P* which designates a bond to theterminal end of an internucleoside linkage to a preceding monomer, or a3′-terminal group selected from hydrogen, hydroxy, optionallysubstituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkylcarbonyloxy,optionally substituted aryloxy, and —W-A′, wherein W is selected from—O—, —S—, and —N(R^(H))— where R^(H) is selected from hydrogen and C₁_(—) ₆-alkyl, and wherein A′ is selected from DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where photochemically activegroups is selected from quinones, diazirines, aromatic azides,benzophenones, psoralens, diazo compounds, and diazirino compounds, andthermochemically active groups is selected from carboxylic acids,carboxylic acid esters, carboxylic acid halides, carboxylic acid azides,carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters,sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes,ketones, primary alcohols, secondary alcohols, tertiary alcohols,phenols, alkyl halides, thiols, disulphides, primary amines, secondaryamines, tertiary amines, hydrazines, epoxides, maleimides, and boronicacid derivatives; the substituents R²* and R⁴* together designates abiradical selected from —CH₂—O—, —NH—CH₂—, —N(CH₃)—CH₂— and —S—CH₂—;each of the substituents R¹*, R², R³, R³*, R⁵, and R⁵* which are notinvolved in P, or P*, designate hydrogen.